Antibodies to CD70

ABSTRACT

The present invention relates to antibodies and antigen binding fragments thereof which bind to the human CD70 protein with high affinity and display potent inhibition of tumour cell growth.

TECHNICAL FIELD

The present invention relates to antibodies and antigen binding fragments thereof which bind to the human CD70 protein with high affinity and display potent inhibition of tumour cell growth.

BACKGROUND

The cytokine receptor CD27 is a member of the tumour necrosis factor receptor (TFNR) superfamily, which play a role in cell growth and differentiation, as well as apoptosis. The ligand for CD27 is CD70, which belongs to the tumour necrosis factor family of ligands. CD70 is a 193 amino acid polypeptide having a 20 amino acid hydrophilic N-terminal domain and a C-terminal domain containing 2 potential N-linked glycosylation sites (Goodwin, R. G. et al. (1993) Cell 73:447-56; Bowman et al. (1994) Immunol 152: 1756-61). Based on these features, CD70 was determined to be a type II transmembrane protein having an extracellular C-terminal portion.

CD70 is transiently found on activated T and B lymphocytes and dendritic cells (Hintzen et al. (1994) J. Immunol. 152: 1762-1773; Oshima et al. (1998) Int. Immunol. 10:517-26; Tesselaar et al. (2003) J. Immunol. 170:33-40). In addition to expression on normal cells, CD70 expression has been reported in different types of cancers including renal cell carcinomas, metastatic breast cancers, brain tumours, leukemias, lymphomas and nasopharyngeal carcinomas (Dunker et al. (2005) J Urol. 173:2150-3; Sloan et al. (2004) Am J Pathol. 164:315-23; Held-Feindt and Mentlein (2002) Int J Cancer 98:352-6; Hishima et al. (2000) Am J Surg Pathol. 24:742-6; Lens et al. (1999) Br J Haematol. 106:491-503). The interaction of CD70 with CD27 has also been proposed to play a role in cell-mediated autoimmune disease and the inhibition of TNF-alpha production (Nakajima et al. (2000) J. Neuroimmunol. 109:188-96).

Accordingly, CD70 represents a target for the treatment of cancer, autoimmune disorders and a variety of other diseases characterized by CD70 expression.

WO 2006/0044643 describes CD70 antibodies containing an antibody effector domain which can mediate one or more of ADCC, ADCP, CDC or ADC and either exert a cytostatic or cytotoxic effect on a CD70-expressing cancer or exert an immunosuppressive effect on a CD70-expressing immunological disorder in the absence of conjugation to a cytostatic or cytotoxic agent. The antibodies exemplified therein are based on the antigen-binding regions of two monoclonal antibodies, denoted 1F6 and 2F2.

WO 2007/038637 describes fully human monoclonal antibodies that bind to CD70. These antibodies are characterised by binding to human CD70 with a K_(D) of 1×10⁻⁷ M or less. The antibodies also bind to, and are internalised by, renal cell carcinoma tumor cell lines which express CD70, such as 786-O.

SUMMARY OF THE INVENTION

Provided herein are antibodies, or antigen binding fragments thereof, (referred to herein as CD70 antibodies) which bind to the human CD70 protein and exhibit properties which are different, and generally superior, to CD70 antibodies described in the prior art. The superior properties of these antibodies are advantageous with regard to use in human therapy, particularly treatment of CD70-expressing cancers and also immunological disorders.

The CD70 antibodies described herein are characterised by extremely high binding affinity for human CD70. All preferred embodiments described herein exhibit a binding affinity for recombinant human CD70 (measured by Biacore™ surface plasmon resonance as described herein) which is significantly higher than the most potent prior art CD70 antibodies proposed for human therapy, including prior art CD70 antibodies of “fully human” origin. In addition, all preferred embodiments of the CD70 antibodies described herein exhibit superior (i.e. higher affinity) binding to CD70 expressed on the surface of human cell lines, specifically human cancer cell lines, when compared to prior art CD70 antibodies proposed for human therapy. This superior binding to cell-surface CD70 is particularly marked in regard to human cancer cell lines which express CD70 at “low copy number” and is of direct relevance to use of the antibodies in human therapy. Still further, preferred embodiments of the CD70 antibodies described herein exhibit substantially improved binding to cancer cells isolated from human patients, particularly cancer cells isolated from patients with chronic lymphocytic leukaemia (CLL), when compared to prior art CD70 antibodies proposed for human therapeutic use.

Therefore, in a first aspect of the invention there is provided an antibody, or an antigen binding fragment thereof, which binds to human CD70, said antibody or antigen binding fragment comprising at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), wherein said VH and VL domain exhibit an off-rate (k_(off) measured by Biacore™) for human CD70 of less than 7×10⁻⁴ s⁻¹, when tested as a Fab fragment using the standard Biacore™ protocol described herein.

In a preferred embodiment the antibody or antigen binding fragment comprises at least one heavy chain variable domain (VH) and at least one light chain variable domain (VL), wherein said VH and VL domain exhibit an off-rate for human CD70 of 5×10⁻⁴ s⁻¹ or less, or 2×10⁻⁴ s⁻¹ or less, or 1×10⁻⁴ s⁻¹ or less. Most preferably the CD70 antibody will exhibit an off-rate for CD70 in the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹, when tested as a Fab fragment.

There is also provided an antibody which binds to human CD70, said antibody comprising two Fab regions, wherein each of the Fab regions binds to human CD70 and exhibits an off-rate for human CD70 of 5×10⁻⁴ s⁻¹ or less, or 2×10⁻⁴ s⁻¹ or less, or 1×10⁻⁴ s⁻¹ or less and preferably in the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹, when tested as a Fab fragment. The two Fab regions may be identical or they may differ in terms of binding properties, e.g. affinity for human CD70. The two Fab regions may bind to the same epitope or overlapping epitopes on human CD70 or they may bind to distinct, non-overlapping epitopes on human CD70. The two Fab regions may differ from one another in terms of amino acid sequence within one or both of the VH and VL domains.

Preferred embodiments of the CD70 antibodies provided herein may, in addition to the extremely high binding affinity for CD70, exhibit potent blocking or inhibition of the interaction between CD70 and its ligand CD27. The preferred CD70 antibodies which exhibit both high affinity binding to CD70 and potent blocking of the CD70/CD27 interaction are particularly advantageous as therapeutic agents for treatment of disease indications where blocking of CD70/CD27 signalling enhances therapeutic efficacy (e.g. in addition to cell-killing mediated by the effector functions of the CD70 antibody), for example autoimmune diseases and cancers which co-express CD70 and CD27.

Not all of the CD70 antibodies described herein exhibit potent blocking of the CD70/CD27 interaction in addition to the high affinity binding to CD70. Also described herein are a number of CD70 antibodies which exhibit very high affinity binding to CD70 but do not show significant blocking of the CD70/CD27 interaction. The properties of these antibodies are described elsewhere herein. The availability of high affinity non-blocking CD70 antibodies may enhance/extend the range of therapeutic possibilities.

Preferred embodiments of the CD70 antibodies described herein, exhibiting very high binding affinity for human CD70, are also characterised by a combination of binding properties which is not exhibited by prior art CD70 antibodies proposed for human therapeutic use. Accordingly, the preferred CD70 antibodies described herein are characterised by:

-   -   (a) binding within the amino acid sequence HIQVTLAICSS (SEQ ID         NO:342) in human CD70;     -   (b) cross-reactivity with CD70 homologs of rhesus macaque         (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus);     -   (c) binding to both native human CD70 and heat denatured         recombinant human CD70.

This combination of binding properties, which is not exhibited by the prior art antibodies proposed for human therapeutic use, indicates binding to a novel epitope on CD70 which is different to the epitopes bound by prior art CD70 antibodies.

The combination of binding properties exhibited by the preferred CD70 antibodies is advantageous in the context of human drug development. In particular, cross-reactivity with simian CD70 homologs enables toxicology studies on CD70 antibodies proposed for human therapeutic use to be carried out in primate models.

The preferred CD70 antibodies described herein still further exhibit favourable properties which are relevant to commercial manufacture as a therapeutic antibody product. As discussed elsewhere herein, the preferred CD70 antibodies provided herein exhibit extremely high level expression in the recombinant expression systems utilised for commercial manufacture of clinical grade therapeutic antibody products. The expression levels achievable with the preferred CD70 antibodies far exceed the levels typically achieved even for “fully human” therapeutic antibody products. In addition, the preferred CD70 antibody products (produced by recombinant expression in a foimat suitable for human therapeutic use) exhibit outstanding thermal stability, which is superior to typical therapeutic antibody products.

The CD70 antibodies provided herein with superior binding affinity for human CD70 and the other advantageous properties listed above are camelid-derived (for example llama-derived). The camelid-derived CD70 antibodies may be isolated or recombinantly expressed monoclonal antibodies. Preferred embodiments may be a humanised (or germlined) monoclonal antibody (e.g. a humanised variant of a camelid-derived antibody), a chimeric antibody (e.g. a camelid-human chimeric antibody) or a humanised chimeric antibody (e.g. a chimeric antibody comprising humanised variants of camelid VH and VL domains and constant domains of a human antibody).

Camelid-derived CD70 antibodies may comprise at least one hypervariable loop or complementarity determining region obtained from a VH domain or a VL domain of a species in the family Camelidae. In a particular embodiment, the CD70 antibody, or antigen binding fragment thereof, may comprise a heavy chain variable domain (VH) and light chain variable domain (VL), wherein the VH and VL domains, or one or more CDRs thereof, are camelid-derived. In particular embodiments the antibody or antigen binding fragment thereof may comprise llama VH and VL domains, or human germlined variants of llama VH and VL domains. This antibody, or antigen binding fragment, may exhibit “high human homology”, as defined herein.

The camelid-derived CD70 antibodies described herein typically exhibit VH and/or VL region amino acid sequences having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity the closest matching human antibody germline sequence.

Further preferred embodiments of the invention include humanised (or human germlined) variants of the camelid-derived CD70 antibodies. In particular, the invention provides humanised or human germlined variants of the llama-derived CD70 antibodies described herein.

In a further aspect of invention there is provided a chimeric camelid-human antibody which binds human CD70, wherein the antigen-binding portions of the antibody (e.g. VH and/or VL domains or CDRs thereof) are camelid-derived and the constant regions of the antibody are derived from a human antibody. In particular, the invention provides a chimeric llama-human antibody which binds human CD70.

In a further aspect of invention there is provided a humanised variant of a chimeric camelid-human antibody which binds human CD70, wherein the antigen-binding portions of the antibody (e.g. VH and/or VL domains or CDRs thereof) are humanised variants of camelid-derived sequences and the constant regions of the antibody are derived from a human antibody. In particular, the invention provides a humanised variant of a chimeric llama-human antibody which binds human CD70.

Preferred (but non-limiting) embodiments of the CD70 antibodies, or antigen binding fragments thereof, are defined below by reference to specific structural characteristics, i.e. specified amino acid sequences of either the CDRs (one or more of SEQ ID Nos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 or 268 (light chain CDR1), or entire variable domains (one or more of SEQ ID NOs: 177-188, 212-223, 274 or 275 (VH) or SEQ ID Nos:189-211, 230-245, 276 or 277 (VL)). All of these antibodies bind to human CD70 with high affinity, exhibiting an off-rate for human CD70 of 5×10⁻⁴ s⁻¹ or less, and typically in the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹, when tested as a Fab fragment.

The invention also provides humanised/germlined variants of these antibodies, plus affinity variants and variants containing conservative amino acid substitutions, as defined herein. Specifically provided are chimeric antibodies containing VH and VL domains which are camelid-derived, or human germlined variants thereof, fused to constant domains of human antibodies, in particular human IgG1, IgG2, IgG3 or IgG4. The heavy chain variable domains defined above can be utilised as single domain antibodies, or may be included within a conventional four-chain antibody or other antigen binding proteins, such as for example Fab, Fab′, F(ab′)2, bi-specific Fab′ s, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, a single chain variable fragment (scFv) and multi specific antibodies.

Preferred embodiments of the CD70 antibodies are antibodies, or antigen binding fragments thereof, comprising a heavy chain variable domain comprising a variable heavy chain CDR3, a variable heavy chain CDR2 and a variable heavy chain CDR1, wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of:

-   SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID     NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ     ID NO:58, SEQ ID NO:59, SEQ ID NO:262 and SEQ ID NO:263, and     sequence variants of any one of the recited sequences, wherein the     sequence variant comprises one, two or three amino acid     substitutions in the recited sequence;

said variable heavy chain CDR2 optionally comprises an amino acid sequence selected from the group consisting of: amino acid sequences of SEQ ID NO: 306 [X₁X₂X₃X₄X₅X₆X₇X₈X₉ YYADSVKX₁₀], wherein

X₁ is any amino acid, preferably D, T, S or E,

X₂ is any amino acid, preferably I,

X₃ is any amino acid, preferably N, S, T or Y,

X₄ is any amino acid, preferably N, M, S or T,

X₅ is any amino acid, preferably E, D, Y or H,

X₆ is any amino acid, preferably G, D, S or N,

X₇ is any amino acid, preferably G, Y, S, D or M,

X₈ is any amino acid, preferably T, E, S, N, Y or R,

X₉ is any amino acid, preferably T, A or R, and

X₁₀ is any amino acid, preferably G or S,

-   and the amino acid sequences of SEQ ID NO:30, SEQ ID NO:32, SEQ ID     NO:33, SEQ ID NO:34, SEQ ID NO:249, SEQ ID NO:258 and SEQ ID NO:259     and sequence variants of any one of the recited sequences, wherein     the sequence variant comprises one, two or three amino acid     substitutions in the recited sequence; and

said variable heavy chain CDR1 optionally comprises an amino acid sequence selected from the group consisting of: amino acid sequences of SEQ ID NO: 307 [X₁ YYMN], wherein

X₁ is any amino acid, preferably V, G or A, amino acid sequences of SEQ ID NO: 308 [X₁X₂ AMS], wherein

X₁ is any amino acid, preferably D, T, S, N or G and

X₂ is any amino acid, preferably Y, S or P,

-   and the amino acid sequences of SEQ ID NO:10, SEQ ID NO:15, SEQ ID     NO:16, SEQ ID NO:248, SEQ ID NO:256 and SEQ ID NO:257 and sequence     variants of any one of the recited sequences, wherein the sequence     variant comprises one, two or three amino acid substitutions in the     recited sequence.

The heavy chain variable domain may comprise any one of the listed variable heavy chain CDR3 sequences (HCDR3) in combination with any one of the variable heavy chain CDR2 sequences (HCDR2) and any one of the variable heavy chain CDR1 sequences (HCDR1). However, certain combinations of HCDR3 and HCDR2 and HCDR1 are particularly preferred, these being the “native” combinations which derive from a single common VH domain. These preferred combinations are listed in Table 6 and Table 14A.

The antibody or antigen binding fragment thereof may additionally comprise a light chain variable domain (VL), which is paired with the VH domain to form an antigen binding domain. Preferred light chain variable domains are those comprising a variable light chain CDR3, a variable light chain CDR2 and a variable light chain CDR1, wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:271 and SEQ ID NO:273 and sequence variants of any one of the recited sequences, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence;

said variable light chain CDR2 optionally comprises an amino acid sequence selected from the group consisting of:

-   (a) amino acid sequences of SEQ ID NO: 310 [X₁ TX₂X₁ RHS], wherein

X₁ is any amino acid, preferably N or S,

X₂ is any amino acid, preferably N, S or A, and

X₃ is any amino acid, preferably S, N or T,

-   (b) amino acid sequences of SEQ ID NO: 311 [YYSDSX₁X₂X₃ QX₄ S],     wherein

X₁ is any amino acid, preferably Y, V or L,

X₂ is any amino acid, preferably K or S,

X₃ is any amino acid, preferably H or N, and

X₄ is any amino acid, preferably G or S,

-   (c) amino acid sequences of SEQ ID NO: 312 [X₁ NX₂ NRPS], wherein

X₁ is any amino acid, preferably V, I or Y, and

X₂ is any amino acid, preferably N or T,

-   (d) amino acid sequences of SEQ ID NO: 313 [GDNX₁X₂ PL], wherein

X₁ is any amino acid, preferably Y, and

X₂ is any amino acid, preferably R or M,

-   (e) amino acid sequences of SEQ ID NO:314 [X₁ DDX₂ RPS], wherein

X₁ is any amino acid, preferably D or G, and

X₂ is any amino acid, preferably S or I,

-   and the amino acid sequences of SEQ ID NO:113, SEQ ID NO:116, SEQ ID     NO:120 and SEQ ID NO:270, and sequence variants of any one of the     recited sequences, wherein the sequence variant comprises one, two     or three amino acid substitutions in the recited sequence; and

said variable light chain CDR1 optionally comprises an amino acid sequence selected from the group consisting of:

-   (a) amino acid sequences of SEQ ID NO:315 [GLX₁ SGSX₂ TX₃X₄X₅ YPX₆],     wherein

X₁ is any amino acid, preferably S or T,

X₂ is any amino acid, preferably V or A,

X₃ is any amino acid, preferably S or T,

X₄ is any amino acid, preferably S, T or G,

X₅ is any amino acid, preferably N or H,

X₆ is any amino acid, preferably G, D or E,

-   (b) amino acid sequences of SEQ ID NO:316 [TLX₁ SX₂X₃X₄X₅ GX₆ YDIS],     wherein

X₁ is any amino acid, preferably S, N or I,

X₂ is any amino acid, preferably G or A,

X₃ is any amino acid, preferably N or D,

X₄ is any amino acid, preferably N or S,

X₅ is any amino acid, preferably V or I,

X₆ is any amino acid, preferably N or S,

-   (c) amino acid sequences of SEQ ID NO:317 [QGGNLX₁ LYGAN], wherein

X₁ is any amino acid, preferably G or W,

-   (d) amino acid sequences of SEQ ID NO:318 [RGDX₁ LX₂X₃ YX₄X₅ N],     wherein

X₁ is any amino acid, preferably S or T,

X₂ is any amino acid, preferably E or R,

X₃ is any amino acid, preferably R or N,

X₄ is any amino acid, preferably G or H,

X₅ is any amino acid, preferably T or A,

-   (e) amino acid sequences of SEQ ID NO:319 [GX₁X₂ SGSVTSX₃NFPT],     wherein

X₁ is any amino acid, preferably V or L,

X₂ is any amino acid, preferably K or T,

X₃ is any amino acid, preferably T or D,

-   and the amino acid sequences of SEQ ID NO:82, SEQ ID NO:87, SEQ ID     NO:88, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO: 252, SEQ ID NO:253,     SEQ ID NO:267 and SEQ ID NO:268 and sequence variants of any one of     the recited sequences, wherein the sequence variant comprises one,     two or three amino acid substitutions in the recited sequence.

The light chain variable domain may comprise any one of the listed variable light chain CDR3 sequences (LCDR3) in combination with any one of the variable light chain CDR2 sequences (LCDR2) and any one of the variable light chain CDR1 sequences (LCDR1). However, certain combinations of LCDR3 and LCDR2 and LCDR1 are particularly preferred, these being the “native” combinations which derive from a single common VL domain. These preferred combinations are listed in Table 7 and Table 15A.

Any given CD70 antibody or antigen binding fragment thereof comprising a VH domain paired with a VL domain to form a binding site for CD70 antigen will comprise a combination of 6 CDRs: variable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1). Although all combinations of 6 CDRs selected from the CDR sequence groups listed above are permissible, and within the scope of the invention, certain combinations of 6 CDRs are particularly preferred; these being the “native” combinations within a single Fab exhibiting high affinity binding to human CD70.

Preferred combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy chain CDR3 (HCDR3), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR1 (HCDR1), variable light chain CDR3 (LCDR3), variable light chain CDR2 (LCDR2) and variable light chain CDR1 (LCDR1) selected from the group consisting of:

-   (i) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:27,     HCDR1 comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:160, LCDR2     comprising SEQ ID NO:119, and LCDR1 comprising SEQ ID NO:250; -   (ii) HCDR3 comprising SEQ ID NO:49, HCDR2 comprising SEQ ID NO:26,     HCDR1 comprising SEQ ID NO:10, LCDR3 comprising SEQ ID NO:148, LCDR2     comprising SEQ ID NO:109, and LCDR1 comprising SEQ ID NO:77; -   (iii) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:27,     HCDR1 comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:149, LCDR2     comprising SEQ ID NO:110, and LCDR1 comprising SEQ ID NO:78; -   (iv) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:28,     HCDR1 comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:150, LCDR2     comprising SEQ ID NO:111, and LCDR1 comprising SEQ ID NO:79; -   (v) HCDR3 comprising SEQ ID NO:50, HCDR2 comprising SEQ ID NO:28,     HCDR1 comprising SEQ ID NO:11, LCDR3 comprising SEQ ID NO:151, LCDR2     comprising SEQ ID NO:110, and LCDR1 comprising SEQ ID NO:80; -   (vi) HCDR3 comprising SEQ ID NO:51, HCDR2 comprising SEQ ID NO:29,     HCDR1 comprising SEQ ID NO:12, LCDR3 comprising SEQ ID NO: 152,     LCDR2 comprising SEQ ID NO:110, and LCDR1 comprising SEQ ID NO:80; -   (vii) HCDR3 comprising SEQ ID NO:52, HCDR2 comprising SEQ ID NO:30,     HCDR1 comprising SEQ ID NO:13, LCDR3 comprising SEQ ID NO:153, LCDR2     comprising SEQ ID NO: 112, and LCDR1 comprising SEQ ID NO:81; -   (viii) HCDR3 comprising SEQ ID NO:53, HCDR2 comprising SEQ ID NO:31,     HCDR1 comprising SEQ ID NO:14, LCDR3 comprising SEQ ID NO:154, LCDR2     comprising SEQ ID NO:113, and LCDR1 comprising SEQ ID NO:82; -   (ix) HCDR3 comprising SEQ ID NO:54, HCDR2 comprising SEQ ID NO:32,     HCDR1 comprising SEQ ID NO:15, LCDR3 comprising SEQ ID NO:155, LCDR2     comprising SEQ ID NO:114, and LCDR1 comprising SEQ ID NO:83; -   (x) HCDR3 comprising SEQ ID NO:55, HCDR2 comprising SEQ ID NO:33,     HCDR1 comprising SEQ ID NO:16, LCDR3 comprising SEQ ID NO:156, LCDR2     comprising SEQ ID NO:115, and LCDR1 comprising SEQ ID NO:84; -   (xi) HCDR3 comprising SEQ ID NO:56, HCDR2 comprising SEQ ID NO:34,     HCDR1 comprising SEQ ID NO:17, LCDR3 comprising SEQ ID NO:157, LCDR2     comprising SEQ ID NO:116, and LCDR1 comprising SEQ ID NO:85; -   (xii) HCDR3 comprising SEQ ID NO:57, HCDR2 comprising SEQ ID NO:35,     HCDR1 comprising SEQ ID NO: 18, LCDR3 comprising SEQ ID NO:158,     LCDR2 comprising SEQ ID NO: 117, and LCDR1 comprising SEQ ID NO: 84; -   (xiii) HCDR3 comprising SEQ ID NO: 58, HCDR2 comprising SEQ ID NO:     36, HCDR1 comprising SEQ ID NO: 19, LCDR3 comprising SEQ ID NO: 159,     LCDR2 comprising SEQ ID NO: 118, and LCDR1 comprising SEQ ID NO: 86; -   (xiv) HCDR3 comprising SEQ ID NO: 50, HCDR2 comprising SEQ ID NO:     27, HCDR1 comprising SEQ ID NO: 11, LCDR3 comprising SEQ ID NO: 161,     LCDR2 comprising SEQ ID NO:120, and LCDR1 comprising SEQ ID NO:88; -   (xv) HCDR3 comprising SEQ ID NO: 50, HCDR2 comprising SEQ ID NO: 27,     HCDR1 comprising SEQ ID NO: 11, LCDR3 comprising SEQ ID NO: 162,     LCDR2 comprising SEQ ID NO: 121, and LCDR1 comprising SEQ ID NO: 89; -   (xvi) HCDR3 comprising SEQ ID NO: 50, HCDR2 comprising SEQ ID NO:     27, HCDR1 comprising SEQ ID NO: 11, LCDR3 comprising SEQ ID NO: 163,     LCDR2 comprising SEQ ID NO: 122, and LCDR1 comprising SEQ ID NO: 90; -   (xvii) HCDR3 comprising SEQ ID NO: 51, HCDR2 comprising SEQ ID NO:     29, HCDR1 comprising SEQ ID NO: 12, LCDR3 comprising SEQ ID NO: 164,     LCDR2 comprising SEQ ID NO: 123, and LCDR1 comprising SEQ ID NO:91; -   (xviii) HCDR3 comprising SEQ ID NO: 51, HCDR2 comprising SEQ ID NO:     29, HCDR1 comprising SEQ ID NO: 12, LCDR3 comprising SEQ ID NO: 164,     LCDR2 comprising SEQ ID NO: 124, and LCDR1 comprising SEQ ID NO: 91; -   (xix) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO:     37, HCDR1 comprising SEQ ID NO: 12, LCDR3 comprising SEQ ID NO: 165,     LCDR2 comprising SEQ ID NO: 125, and LCDR1 comprising SEQ ID NO: 92; -   (xx) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO: 37,     HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 165,     LCDR2 comprising SEQ ID NO: 126, and LCDR1 comprising SEQ ID NO: 93; -   (xxi) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO:     37, HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 166,     LCDR2 comprising SEQ ID NO: 127, and LCDR1 comprising SEQ ID NO: 92; -   (xxii) HCDR3 comprising SEQ ID NO:59, HCDR2 comprising SEQ ID NO:     37, HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 167,     LCDR2 comprising SEQ ID NO: 128, and LCDR1 comprising SEQ ID NO: 94; -   (xxiii) HCDR3 comprising SEQ ID NO: 59, HCDR2 comprising SEQ ID NO:     37, HCDR1 comprising SEQ ID NO: 20, LCDR3 comprising SEQ ID NO: 168,     LCDR2 comprising SEQ ID NO: 110, and LCDR1 comprising SEQ ID NO: 95; -   (xxiv) HCDR3 comprising SEQ ID NO: 262, HCDR2 comprising SEQ ID NO:     258, HCDR1 comprising SEQ ID NO: 256, LCDR3 comprising SEQ ID NO:     271, LCDR2 comprising SEQ ID NO: 110, and LCDR1 comprising SEQ ID     NO: 267; -   (xxv) HCDR3 comprising SEQ ID NO: 263, HCDR2 comprising SEQ ID NO:     259, HCDR1 comprising SEQ ID NO: 257, LCDR3 comprising SEQ ID NO:     273, LCDR2 comprising SEQ ID NO: 270, and LCDR1 comprising SEQ ID     NO: 268.

Further preferred CD70 antibodies, exhibiting high affinity binding to human CD70, include isolated antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:274 and SEQ ID NO:275 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity to one of the recited sequences, and optionally comprising a light chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:223, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:276 and SEQ ID NO:277 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity to one of the recited sequences.

Although all possible pairings of VH domains and VL domains selected from the VH and VL domain sequence groups listed above are permissible, and within the scope of the invention, certain combinations VH and VL are particularly preferred; these being the “native” combinations within a single Fab exhibiting high affinity binding to human CD70. Accordingly, preferred CD70 antibodies, or antigen binding fragments thereof, exhibiting high affinity CD70 binding are those comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the combination is selected from the group consisting of:

-   (i) a heavy chain variable domain comprising the amino acid sequence     of SEQ ID NO:223 and a light chain variable domain comprising the     amino acid sequence of SEQ ID NO:241; -   (ii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:177 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:189; -   (iii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:178 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO: 190; -   (iv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:179 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:191; -   (v) a heavy chain variable domain comprising the amino acid sequence     of SEQ ID NO:179 and a light chain variable domain comprising the     amino acid sequence of SEQ ID NO:192; -   (vi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:180 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:193; -   (vii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:181 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:194; -   (viii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:182 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:195; -   (ix) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:183 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:196; -   (x) a heavy chain variable domain comprising the amino acid sequence     of SEQ ID NO:184 and a light chain variable domain comprising the     amino acid sequence of SEQ ID NO:197; -   (xi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:185 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:198; -   (xii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:186 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:199; -   (xiii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:187 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:200; -   (xiv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:178 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:201; -   (xv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:178 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:202; -   (xvi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:178 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:203; -   (xvii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:178 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:204; -   (xviii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:180 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:205; -   (xix) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:180 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:206; -   (xx) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:188 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:207; -   (xxi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:188 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:208; -   (xxii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:188 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:209; -   (xxiii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:188 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:210; -   (xxiv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:188 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:211; -   (xxv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:274 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:276; -   (xxvi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:275 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:277.

For each of the specific VH/VL combinations listed above, it is also permissible, and within the scope of the invention, to combine a VH domain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to the recited VH domain sequence with a VL domain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to the recited VL domain sequence.

In the preceding paragraph, and elsewhere herein, the structure of the antibodies/antigen binding fragments is defined on the basis of % sequence identity with a recited reference sequence (with a given SEQ ID NO). In this context, % sequence identity between two amino acid sequences may be determined by comparing these two sequences aligned in an optimum manner and in which the amino acid sequence to be compared can comprise additions or deletions with respect to the reference sequence for an optimum alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 in order to obtain the percentage of identity between these two sequences. Typically, the comparison window with correspond to the full length of the sequence being compared. For example, it is possible to use the BLAST program, “BLAST 2 sequences” (Tatusova et al, “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250) available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penalty”: 5, and “extension gap penalty”: 2; the matrix chosen being, for example, the matrix “BLOSUM 62” proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.

The most preferred CD70 antibodies provided herein, which exhibit a particularly advantageous combination of properties, including extremely high affinity binding to human CD70, are those based on the antigen-binding portion of the llama-derived Fab denoted 27B3 in the accompanying examples, plus human germlined variants of 27B3, including the germlined variants identified in the accompanying examples. 27B3 and its germlined variants, particularly variants based on the CDRs or complete variable domains of variant 41D12, exhibit an extremely advantageous combination of properties, summarised as follows: high affinity binding to recombinant human CD70, strong binding to cell surface CD70, specifically binding to CD70 expressed on cancer cell lines, particularly cell lines which express CD70 at “low copy number” and strong binding to cancer cells isolated from patient samples (CLL), potent blocking of the CD70/CD27 interaction, potent effector function—particularly when expressed as a chimera with human IgG1 constant regions, and especially when expressed as a non-fucosylated IgG1, cross-reactivity with CD70 homologs of rhesus macaque and cynomolgus monkey enabling toxicology studies in primate species, binding to both native (i.e. cell-surface) and heat denatured CD70, partial or low levels of internalisation on certain cancer cell lines. All of these characteristics in combination render 41D12, and indeed other 27B3 variants and the other CD70 antibodies described herein which exhibit similar properties, an outstanding candidate for therapeutic use in the treatment of CD70-associated diseases, specifically CD70-expressing cancers and immunological disorders.

27B3, and its variants, are characterised by the presence of the heavy chain variable CDR3 sequence shown as SEQ ID NO:50 (DAGYSNHVPIFDS).

Accordingly, preferred embodiments of the CD70 antibody, or antigen binding fragments thereof, are those comprising a heavy chain variable domain wherein the variable heavy chain CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:50 or a sequence variant thereof, wherein the sequence variant comprises one, two or three amino acid substitutions in the recited sequence.

More preferred embodiments are antibodies or antigen binding fragments thereof which include the same combination of heavy chain CDRs as 27B3, or human germlined variants of 27B3. Accordingly, the antibody or antigen binding fragment thereof, may comprise a heavy chain variable domain wherein

the variable heavy chain CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:50 or a sequence variant thereof;

the variable heavy chain CDR2 comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:27, SEQ ID NO:249 and sequence variants thereof; and

the variable heavy chain CDR1 comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:11, SEQ ID NO:248 and sequence variants thereof, wherein the sequence variants comprise one, two or three amino acid substitutions (e.g., conservative substitutions, humanising substitutions or affinity variants) in the recited sequence.

Any combination of HCDR3, HCDR2 and HCDR1 from within the recited groups of CDR sequences is permissible, and within the scope of the invention, however certain combinations are particular preferred. Accordingly, in preferred embodiments the antigen or antigen binding fragment thereof may comprise a heavy chain variable domain wherein the combination of HCDR3, HCDR2 and HCDR1 is selected from the following:

-   -   (a) the variable heavy chain CDR3 comprises or consists of SEQ         ID NO:50 (DAGYSNHVPIFDS) or a sequence variant thereof;         -   the variable heavy chain CDR2 comprises or consists of SEQ             ID NO:27 (DINNEGGTTYYADSVKG) or a sequence variant thereof;             and         -   the variable heavy chain CDR1 comprises or consists of SEQ             ID NO:11 (VYYMN) or a sequence variant thereof,         -   wherein the sequence variants comprises one, two or three             amino acid sub stitutions (e.g., conservative substitutions,             humanising substitutions or affinity variants) in the             recited sequence;     -   (b) the variable heavy chain CDR3 comprises or consists of SEQ         ID NO:50 (RDAGYSNHVPIFDS) or a sequence variant thereof;         -   the variable heavy chain CDR2 comprises or consists of SEQ             ID NO:249 (DINNEGGATYYADSVKG) or a sequence variant thereof;             and         -   the variable heavy chain CDR1 comprises or consists of SEQ             ID NO:11 (VYYMN) or a sequence variant thereof,         -   wherein the sequence variants comprises one, two or three             amino acid sub stitutions (e.g., conservative substitutions,             humanising substitutions or affinity variants) in the             recited sequence.     -   (c) the variable heavy chain CDR3 comprises or consists of SEQ         ID NO:50 (DAGYSNHVPIFDS) or a sequence variant thereof;         -   the variable heavy chain CDR2 comprises or consists of SEQ             ID NO:27 (DINNEGGTTYYADSVKG) or a sequence variant thereof;             and         -   the variable heavy chain CDR1 comprises or consists of SEQ             ID NO:248 (GYYMN) or a sequence variant thereof,         -   wherein the sequence variants comprises one, two or three             amino acid substitutions (e.g., conservative substitutions,             humanising substitutions or affinity variants) in the             recited sequence.

In preferred embodiments, the antibody or antigen binding fragment thereof also includes a light chain variable domain (VL), paired with the heavy chain variable domain, In the preferred light chain variable domains, the variable light chain CDR3 comprises or consists of SEQ ID NO:160 or a sequence variant thereof;

the variable light chain CDR2 comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:119, SEQ ID NO:110 and sequence variants of the recited sequences; and

the variable light chain CDR1 comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:87, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, SEQ ID NO:253 and sequence variants of the recited sequences, and

wherein the sequence variants comprise one, two or three amino acid substitutions (e.g., conservative substitutions, humanising substitutions or affinity variants) in the recited sequences.

Any combination of LCDR3, LCDR2 and LCDR1 from within the recited groups of light chain CDR sequences is permissible, and within the scope of the invention, however certain combinations are particular preferred. Accordingly, in preferred embodiments the antigen or antigen binding fragment thereof may comprise a light chain variable domain wherein the combination of LCDR3, LCDR2 and LCDR1 is selected from the following:

-   -   (a) the variable light chain CDR3 comprises or consists of SEQ         ID NO:160 (ALFISNPSVE) or a sequence variant thereof;         -   the variable light chain CDR2 comprises or consists of SEQ             TD NO:119 (NTNTRHS) or a sequence variant thereof; and         -   the variable light chain CDR1 comprises or consists of SEQ             ID NO:250 (GLKSGSVTSDNFPT) or a sequence variant thereof,         -   wherein the sequence variants comprises one, two or three             amino acid substitutions (e.g., conservative substitutions,             humanising substitutions or affinity variants) in the             recited sequence;     -   (b) the variable light chain CDR3 comprises or consists of SEQ         ID NO:160 (ALFISNPSVE) or a sequence variant thereof;         -   the variable light chain CDR2 comprises or consists of SEQ             ID NO:119 (NTNTRHS) or a sequence variant thereof; and         -   the variable light chain CDR1 comprises or consists of SEQ             ID NO:87 (GLKSGSVTSTNFPT) or a sequence variant thereof,         -   wherein the sequence variants comprises one, two or three             amino acid substitutions (e.g., conservative substitutions,             humanising substitutions or affinity variants) in the             recited sequence.

Other preferred light chain CDR combinations for human “germlined” variants of 27B3 are given in Table 15A.

The most preferred embodiment of the CD70 antibody or antigen binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) wherein the combination of 6 CDRs which forms the binding site for human CD70 is as follows:

the variable heavy chain CDR3 comprises or consists of SEQ ID NO:50 (DAGYSNHVPIFDS) or a sequence variant thereof;

the variable heavy chain CDR2 comprises or consists of SEQ ID NO:27 (DINNEGGTTYYADSVKG) or a sequence variant thereof;

the variable heavy chain CDR1 comprises or consists of SEQ ID NO:11 (VYYMN) or a sequence variant thereof;

the variable light chain CDR3 comprises or consists of SEQ ID NO:160 (ALFISNPSVE) or a sequence variant thereof;

the variable light chain CDR2 comprises or consists of SEQ ID NO:119 (NTNTRHS) or a sequence variant thereof; and

the variable light chain CDR1 comprises or consists of SEQ ID NO:250 (GLKSGSVTSDNFPT) or a sequence variant thereof,

wherein the sequence variants comprises one, two or three amino acid substitutions (e.g., conservative substitutions, humanising substitutions or affinity variants) in the recited sequence.

Other preferred combinations of 6 CDRs are those “native” combinations which occur in the human germlined variants of 27B3 listed in Tables 14A (heavy chains) and 15A (light chains).

In the foregoing preferred embodiments based on 27B3 and its germlined variants, the antibody preferably includes the CH1 domain, hinge region, CH2 domain and CH3 domain of a human antibody, in particular human IgG1, IgG2, IgG3 or IgG4. The most preferred embodiment is a human IgG1. It is still further preferred for the human IgG1 to be engineering to maximise effector function in one or more of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or antibody-dependent cellular phagocytosis (ADCP). Particularly preferred is a non-fucosylated human IgG1, e.g. a non-fucosylated IgG1 produced using the Potelligent™ technology of BioWa Inc.

Further preferred CD70 antibodies, exhibiting high affinity binding to human CD70, based on the Fab denoted 27B3 and human germlined variants of 27B3, include isolated antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:178, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:225, SEQ ID NO:226, SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:274 and SEQ ID NO:275 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity to one of the recited sequences, and optionally comprising a light chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:201, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:233, SEQ ID NO:234, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, SEQ ID NO:244, SEQ ID NO:245, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:276 and SEQ ID NO:277 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98% or 99% sequence identity to one of the recited sequences.

Although all possible pairings of VH domains and VL domains selected from the VH and VL domain sequence groups listed above are permissible, and within the scope of the invention, certain combinations VH and VL are particularly preferred; these being the “native” combinations within a single Fab exhibiting high affinity binding to human CD70. In the case of the germlined variants of 27B3 recited in Tables 14B and 15B, it may be preferred to retain the original VH/VL pairing Accordingly, preferred CD70 antibodies, or antigen binding fragments thereof, exhibiting high affinity CD70 binding are those comprising a combination of a heavy chain variable domain and a light chain variable domain, wherein the combination is selected from the group consisting of:

-   (i) a heavy chain variable domain comprising the amino acid sequence     of SEQ ID NO:223 and a light chain variable domain comprising the     amino acid sequence of SEQ ID NO:241; -   (ii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:178 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO: 190; -   (iii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:212 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO: 230 -   (iv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:213 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:231; -   (v) a heavy chain variable domain comprising the amino acid sequence     of SEQ ID NO:214 and a light chain variable domain comprising the     amino acid sequence of SEQ ID NO:232; -   (vi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:215 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:235; -   (vii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:216 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:234; -   (viii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:217 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:235; -   (ix) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:218 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:236; -   (x) a heavy chain variable domain comprising the amino acid sequence     of SEQ ID NO:219 and a light chain variable domain comprising the     amino acid sequence of SEQ ID NO:237; -   (xi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:220 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:238; -   (xii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:221 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:239; -   (xiii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:222 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:240; -   (xiv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:224 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:242; -   (xv) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:225 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:243; -   (xvi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:226 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:244; -   (xvii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:227 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:245; -   (xviii) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:228 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:246; -   (xix) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:229 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:247; -   (xx) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:223 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:244; -   (xxi) a heavy chain variable domain comprising the amino acid     sequence of SEQ ID NO:223 and a light chain variable domain     comprising the amino acid sequence of SEQ ID NO:245.

For each of the specific VH/VL combinations listed above, it is also permissible, and within the scope of the invention, to combine a VH domain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to the recited VH domain sequence with a VL domain having an amino acid sequence at least 90%, 92%, 95%, 97% or 99% identical to the recited VL domain sequence.

The most preferred embodiment is a CD70 antibody or antigen binding fragment thereof based on the native VH/VL combination of the human germlined variant denoted 41D12. Accordingly, there is also provided herein an antibody or antigen binding fragment thereof comprising a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence selected from the group consisting of: the amino acid sequence shown as SEQ ID NO:223, germlined variants and affinity variants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or 99% identical thereto, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence selected from the group consisting of: the amino acid sequence shown as SEQ ID NO:241, germlined variants and affinity variants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or 99% identical thereto.

Embodiments wherein the amino acid sequence of the VH domain exhibits less than 100% sequence identity with the sequence shown as SEQ ID NO: 223 may nevertheless comprise heavy chain CDRs which are identical to HCDR1, HCDR2 and HCDR3 of SEQ ID NO:223 (SEQ ID NOs:11, 27 and 50, respectively) whilst exhibiting amino acid sequence variation within the framework regions. Likewise, embodiments wherein the amino acid sequence of the VL domain exhibits less than 100% sequence identity with the sequence shown as SEQ ID NO: 241 may nevertheless comprise heavy chain CDRs which are identical to LCDR1, LCDR2 and LCDR3 of SEQ ID NO:241 (SEQ ID NOs:250, 116 and 160, respectively) whilst exhibiting amino acid sequence variation within the framework regions.

In the foregoing preferred embodiments based on the VH and VL domains of 41D12, or variants thereof, the antibody preferably includes the CH1 domain, hinge region, CH2 domain and CH3 domain of a human antibody, in particular human IgG1, IgG2, IgG3 or IgG4. The most preferred embodiment is a human IgG1. It is still further preferred for the human IgG1 to be engineering to maximise effector function in one or more of ADCC, CDC or ADCP. Particularly preferred is a de-fucosylated human IgG1, preferably prepared using the Potelligent™ expression system.

Another advantageous embodiment is an antibody or antigen binding fragment thereof comprising a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence selected from the group consisting of: the amino acid sequence shown as SEQ ID NO:225, germlined variants and affinity variants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or 99% identical thereto, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence selected from the group consisting of: the amino acid sequence shown as SEQ ID NO:243, germlined variants and affinity variants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or 99% identical thereto; or

Another advantageous embodiment is an antibody or antigen binding fragment thereof comprising a heavy chain variable domain (VH) comprising or consisting of an amino acid sequence selected from the group consisting of: the amino acid sequence shown as SEQ ID NO:226, germlined variants and affinity variants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or 99% identical thereto, and a light chain variable domain (VL) comprising or consisting of an amino acid sequence selected from the group consisting of: the amino acid sequence shown as SEQ ID NO:244, germlined variants and affinity variants thereof and amino acid sequences at least 90%, 95%, 97%, 98% or 99% identical thereto.

Features/Properties of CD70 Antibodies

In the aforementioned aspects and embodiments, the CD70 antibodies, or antigen binding fragments thereof, may each exhibit one or more, or any combination, of the following properties or features:

The antibody or antigen binding fragment may bind to human CD70 with high affinity, exhibiting an off-rate for human CD70 of 7×10⁻⁴ s⁻¹ or less, preferably 5×10⁻⁴ s⁻¹ or less, and typically in the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹, when tested as a Fab fragment.

The antibody or antigen binding fragment may bind to human CD70 with high affinity and inhibit the interaction between CD70 and CD27. Alternatively, the antibody or antigen binding fragment may bind to human CD70 but not inhibit the interaction between CD70 and CD27.

The antibody or antigen binding fragment may bind with high affinity to human CD70 on the surface of CD70-expressing cells.

The antibody or antigen binding fragment may bind to human CD70 on the surface of CD70-expressing cells and be slowly or only partially internalised. A key aspect of the invention is the observation that the CD70 antibodies are in fact very poorly internalised on a large number of CD70-expressing cell-lines, including many CD70-expressing cancer cell lines. This observation is in direct contrast to previous published reports that CD70 antibodies are rapidly internalised following binding to renal cell carcinoma cell lines (see Adam et al., British Journal of Cancer (2006) 95: 298-306; and WO 2007/038637) and has direct implications for therapeutic use of the antibodies. The observation that the CD70 antibodies are very poorly internalised following binding to cancer cells strongly supports the conclusion that therapeutic strategies for treatment of many CD70-expressing cancers, and indeed CD70-associated immunological diseases, should be based on extreme high affinity binding to CD70, coupled with antibody effector function, in particular any one or more of ADCC, CDC or ADCP, and not on the use of immunoconjugates in which the CD70 antibody is linked to a therapeutic agent, e.g. a cytotoxic or cytostatic drug moiety.

The antibody or antigen binding fragment may bind within the amino acid sequence HIQVTLAICSS (SEQ ID NO:342) in human CD70;

The antibody or antigen binding fragment may exhibit cross-reactivity with CD70 of simian origin, specifically the CD70 homologs of rhesus macaque (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus).

The antibody or antigen binding fragment may bind to both native human CD70 (e.g. CD70 expressed on the surface of a cell, such as a cell line or a CD70-expressing cell isolated from a human patient) and heat denatured recombinant human CD70.

The antibody or antigen binding fragment may provide very high production yields (>4 g/L) in recombinant antibody expression systems, such as for example the CHK1SV cell line (proprietary to BioWa/Lonza), as compared to a 1-2 g/L historical average for therapeutic antibody products, resulting in a substantial reduction in production costs.

The antibody or antigen binding fragment may be highly stable under 37° C. storage conditions and in freeze-thaw cycles, which is also a major cost reduction factor.

The antibody may exhibit one or more effector functions selected from antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated phagocytosis (ADCP) against cells expressing human CD70 protein on the cell surface.

The antibody may exhibit ADCC against CD70-expressing cells, e.g. cancer cells or other malignant cells, or immune cells.

The antibody may exhibit enhanced ADCC function in comparison to a reference antibody which is an equivalent antibody comprising a native human Fc domain. In a non-limiting embodiment, the ADCC function may be at least 10× enhanced in comparison to the reference antibody comprising a native human Fc domain. In this context “equivalent” may be taken to mean that the antibody with enhanced ADCC function displays substantially identical antigen-binding specificity and/or shares identical amino acid sequence with the reference antibody, except for any modifications made (relative to native human Fc) for the purposes of enhancing ADCC.

The antibody or antigen binding fragment may inhibit tumour growth in an in vivo tumour xenograft model, in the absence of conjugation to a cytotoxic or cytostatic agent. In a non-limiting embodiment, the inhibition of tumour growth function may be at least 10 fold enhanced in comparison to the reference antibody SGN70.

The antibody or antigen binding fragment may induce apoptosis of CD70-expressing cells.

The antibody may contain the hinge region, CH2 domain and CH3 domain of a human IgG, most preferably human IgG1, IgG2, IgG3 or IgG4.

The antibody may include modifications in the Fc region, such as modifications which enhance antibody effector function as explained elsewhere herein. In particular, the antibody may be a non-fucosylated IgG.

In further aspects, the invention also provides polynucleotide molecules which encode the above-listed CD70 antibodies and antigen binding fragments thereof, in addition to expression vectors comprising the polynucleotides, host cells containing the vectors and methods of recombinant expression/production of the CD70 antibodies.

In a still further aspect, the invention provides a pharmaceutical composition comprising any one of the CD70 antibodies described above and a pharmaceutically acceptable carrier or excipient.

A still further aspect of the invention concerns methods of medical treatment using the above-listed CD70 antibodies, particularly in the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be further understood with reference to the following experimental examples and the accompanying Figures in which:

FIG. 1: Shows the immune response tested in ELISA on recombinant CD70 for llamas immunised with 786-O cells (top) and with Raji cells (bottom).

FIG. 2: illustrates inhibition of binding of CD27 to CD70 by llama-derived CD70 Fabs and reference CD70 Fabs, measured by ELISA.

FIG. 3: is a graphical representation of the signal for llama-derived Fabs tested in a binding Elisa (black) or in an inhibition Elisa (white).

FIG. 4: illustrates inhibition of binding of CD27 to human CD70 (A&B) or rhesus CD70 (C) by chimeric llama-human CD70 mAhs and reference CD70 mAbs measured by ELISA.

FIG. 5: shows binding of chimeric llama-human CD70 mAbs to 786-O cells (A) or MHH-PREB-1 cells (B) as demonstrated by FACS analysis.

FIG. 6: shows inhibition by CD70 specific chimeric llama-human mAbs in a Raji cell based co-culture potency assay.

FIG. 7: shows the results of standard Cr⁵¹ release ADCC assay on 786-O cells.

FIG. 8: shows the results of CDC assay on U266 cells in the presence of 9% human serum.

FIG. 9: demonstrates the efficacy of chimeric llama-human CD70 mAbs in a ADCP assay on 786-O cells

FIG. 10. illustrates antibody internalisation, assessed as MFI OUT for different chimeric llama-human CD70 mAbs as a function of time on 786-O cells in two independent experiments.

FIG. 11: demonstrates survival of mice in a Raji Xenograft model after treatment with chimeric llama-human CD70 mAb 41D12, isotype control and Fc-dead control.

FIG. 12: depicts alignments of the VH and VL amino acid sequences of clone 27B3 with the amino acid sequences of human germline gene segments VH3-38 and VL8-61, respectively.

FIG. 13: shows gelfiltration analysis of samples of germlined 27B3 mAb variants taken after 5 weeks incubation at 37° C.

FIG. 14: shows the potency in CD70 binding, as measured using Biacore, of samples of germlined CD70 mAbs taken at various time points after incubation at various temperatures.

FIG. 15: shows the binding affinity of CD70 mAbs for CD70-expressing cancer cell lines.

FIG. 16: shows the affinity of CD70 mAbs for CLL patient cells expressing CD70.

FIG. 17: shows lysis of SU-DHL-6 cells bound by CD70 mAbs.

FIG. 18: shows inhibition of binding of CD27 to CD70 by CD70 mAbs, measured by ELISA.

FIG. 19: shows an alignment of CD70 sequences from different species.

FIG. 20: shows the binding affinity of CD70 mAbs for human U266 cells, rhesus monkey LCL8864 cells and cynomologus monkey HSC-F cells.

FIG. 21: shows inhibition of binding of CD27 to CD70 of human, rhesus monkey and cynomologus monkey by CD70 mAbs as determined by ELISA.

FIG. 22: shows binding of CD70 mAbs to denatured recombinant CD70, assessed by ELISA.

FIG. 23: shows CD70 chimeric sequences used for epitope mapping.

FIG. 24: illustrates antibody internalisation for CD70 mAbs as a function of time on 786-O cells.

DEFINITIONS

-   “Antibody” or “Immunoglobulin”—As used herein, the term     “immunoglobulin” includes a polypeptide having a combination of two     heavy and two light chains whether or not it possesses any relevant     specific immunoreactivity. “Antibodies” refers to such assemblies     which have significant known specific immunoreactive activity to an     antigen of interest (e.g. human CD70). The term “CD70 antibodies” is     used herein to refer to antibodies which exhibit immunological     specificity for human CD70 protein. As explained elsewhere herein,     “specificity” for human CD70 does not exclude cross-reaction with     species homologues of CD70. Antibodies and immunoglobulins comprise     light and heavy chains, with or without an interchain covalent     linkage between them. Basic immunoglobulin structures in vertebrate     systems are relatively well understood.

The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. All five classes of antibodies are within the scope of the present invention, the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

The light chains of an antibody are classified as either kappa or lambda (κ, λ). Each heavy chain class may be hound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (γ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA, IgD or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.

As indicated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region that defines a three dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the VH and VL chains.

-   “CD70 protein” or “CD70 antigen”—As used herein, the terms “CD70     protein” or “CD70 antigen” or “CD70” are used interchangeably and     refer to a member of the TNF ligand family which is a ligand for     TNFRSF27/CD27. The terms “human CD70 protein” or “human CD70     antigen” or “human CD70” are used interchangeably to refer     specifically to the human homolog, including the native human CD70     protein naturally expressed in the human body and/or on the surface     of cultured human cell lines, as well as recombinant forms and     fragments thereof. Specific examples of human CD70 include the     polypeptide having the amino acid sequence shown under NCBI     Reference Sequence Accession No. NP_001243, or the extracellular     domain thereof. -   “Binding Site”—As used herein, the term “binding site” comprises a     region of a polypeptide which is responsible for selectively binding     to a target antigen of interest (e.g. human CD70). Binding domains     comprise at least one binding site. Exemplary binding domains     include an antibody variable domain. The antibody molecules of the     invention may comprise a single binding site or multiple (e.g., two,     three or four) binding sites. -   “Derived From”—As used herein the term “derived from” a designated     protein (e.g. a CD70 antibody or antigen-binding fragment thereof)     refers to the origin of the polypeptide. In one embodiment, the     polypeptide or amino acid sequence which is derived from a     particular starting polypeptide is a CDR sequence or sequence     related thereto. In one embodiment, the amino acid sequence which is     derived from a particular starting polypeptide is not contiguous.     For example, in one embodiment, one, two, three, four, five, or six     CDRs are derived from a starting antibody. In one embodiment, the     polypeptide or amino acid sequence which is derived from a     particular starting polypeptide or amino acid sequence has an amino     acid sequence that is essentially identical to that of the starting     sequence, or a portion thereof wherein the portion consists of at     least of at least 3-5 amino acids, 5-10 amino acids, at least 10-20     amino acids, at least 20-30 amino acids, or at least 30-50 amino     acids, or which is otherwise identifiable to one of ordinary skill     in the art as having its origin in the starting sequence. In one     embodiment, the one or more CDR sequences derived from the starting     antibody are altered to produce variant CDR sequences, e.g. affinity     variants, wherein the variant CDR sequences maintain CD70 binding     activity. -   “Camelid-Derived”—In certain preferred embodiments, the CD70     antibody molecules of the invention comprise framework amino acid     sequences and/or CDR amino acid sequences derived from a camelid     conventional antibody raised by active immunisation of a camelid     with CD70 antigen. However, CD70 antibodies comprising     camelid-derived amino acid sequences may be engineered to comprise     framework and/or constant region sequences derived from a human     amino acid sequence (i.e. a human antibody) or other non-camelid     mammalian species. For example, a human or non-human primate     framework region, heavy chain portion, and/or hinge portion may be     included in the subject CD70 antibodies. In one embodiment, one or     more non-camelid amino acids may be present in the framework region     of a “camelid-derived” CD70 antibody, e.g., a camelid framework     amino acid sequence may comprise one or more amino acid mutations in     which the corresponding human or non-human primate amino acid     residue is present. Moreover, camelid-derived VH and VL domains, or     humanised variants thereof, may be linked to the constant domains of     human antibodies to produce a chimeric molecule, as extensively     described elsewhere herein. -   “Conservative amino acid substitution”—A “conservative amino acid     substitution” is one in which the amino acid residue is replaced     with an amino acid residue having a similar side chain. Families of     amino acid residues having similar side chains have been defined in     the art, including basic side chains (e.g., lysine, arginine,     histidine), acidic side chains (e.g., aspartic acid, glutamic acid),     uncharged polar side chains (e.g., glycine, asparagine, glutamine,     serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,     alanine, valine, leucine, isoleucine, proline, phenylalanine,     methionine, tryptophan), beta-branched side chains (e.g., threonine,     valine, isoleucine) and aromatic side chains (e.g., tyrosine,     phenylalanine, tryptophan, histidine). Thus, a nonessential amino     acid residue in an immunoglobulin polypeptide may be replaced with     another amino acid residue from the same side chain family. In     another embodiment, a string of amino acids can be replaced with a     structurally similar string that differs in order and/or composition     of side chain family members. -   “Heavy chain portion”—As used herein, the term “heavy chain portion”     includes amino acid sequences derived from the constant domains of     an immunoglobulin heavy chain. A polypeptide comprising a heavy     chain portion comprises at least one of: a CH1 domain, a hinge     (e.g., upper, middle, and/or lower hinge region) domain, a CH2     domain, a CH3 domain, or a variant or fragment thereof. In one     embodiment, an antibody or antigen binding fragment of the invention     may comprise the Fc portion of an immunoglobulin heavy chain (e.g.,     a hinge portion, a CH2 domain, and a CH3 domain). In another     embodiment, an antibody or antigen binding fragment of the invention     may lack at least a portion of a constant domain (e.g., all or part     of a CH2 domain). In certain embodiments, at least one, and     preferably all, of the constant domains are derived from a human     immunoglobulin heavy chain. For example, in one preferred     embodiment, the heavy chain portion comprises a fully human hinge     domain. In other preferred embodiments, the heavy chain portion     comprising a fully human Fc portion (e.g., hinge, CH2 and CH3 domain     sequences from a human immunoglobulin).

In certain embodiments, the constituent constant domains of the heavy chain portion are from different immunoglobulin molecules. For example, a heavy chain portion of a polypeptide may comprise a CH2 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, the constant domains are chimeric domains comprising portions of different immunoglobulin molecules. For example, a hinge may comprise a first portion from an IgG1 molecule and a second portion from an IgG3 or IgG4 molecule. As set forth above, it will be understood by one of ordinary skill in the art that the constant domains of the heavy chain portion may be modified such that they vary in amino acid sequence from the naturally occurring (wild-type) immunoglobulin molecule. That is, the polypeptides of the invention disclosed herein may comprise alterations or modifications to one or more of the heavy chain constant domains (CH1, hinge, CH2 or CH3) and/or to the light chain constant region domain (CL). Exemplary modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.

-   “Chimeric”—A “chimeric” protein comprises a first amino acid     sequence linked to a second amino acid sequence with which it is not     naturally linked in nature. The amino acid sequences may normally     exist in separate proteins that are brought together in the fusion     polypeptide or they may normally exist in the same protein but are     placed in a new arrangement in the fusion polypeptide. A chimeric     protein may be created, for example, by chemical synthesis, or by     creating and translating a polynucleotide in which the peptide     regions are encoded in the desired relationship. Exemplary chimeric     CD70 antibodies include fusion proteins comprising camelid-derived     VH and VL domains, or humanised variants thereof, fused to the     constant domains of a human antibody, e.g. human IgG1, IgG2, IgG3 or     IgG4. -   “Variable region” or “variable domain”—The terms “variable region”     and “variable domain” are used herein interchangeable and are     intended to have equivalent meaning. The term “variable” refers to     the fact that certain portions of the variable domains VH and VL     differ extensively in sequence among antibodies and are used in the     binding and specificity of each particular antibody for its target     antigen. However, the variability is not evenly distributed     throughout the variable domains of antibodies. It is concentrated in     three segments called “hypervariable loops” in each of the VL domain     and the VH domain which form part of the antigen binding site. The     first, second and third hypervariable loops of the VLambda light     chain domain are referred to herein as L1(λ), L2(λ) and L3(λ) and     may be defined as comprising residues 24-33 (L1(λ), consisting of 9,     10 or 11 amino acid residues), 49-53 (L2(λ), consisting of 3     residues) and 90-96 (L3(λ), consisting of 5 residues) in the VL     domain (Morea et al., Methods 20:267-279 (2000)). The first, second     and third hypervariable loops of the VKappa light chain domain are     referred to herein as L1 (κ), L2(κ) and L3(κ) and may be defined as     comprising residues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or     13 residues), 49-53 (L2(κ), consisting of 3 residues) and 90-97     (L3(κ), consisting of 6 residues) in the VL domain (Morea et al.,     Methods 20:267-279 (2000)). The first, second and third     hypervariable loops of the VH domain are referred to herein as H1,     H2 and H3 and may be defined as comprising residues 25-33 (H1,     consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4     residues) and 91-105 (H3, highly variable in length) in the VH     domain (Morea et al., Methods 20:267-279 (2000)).

Unless otherwise indicated, the terms L1, L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both Vkappa and Vlambda isotypes. The terms H1, H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise part of a “complementarity determining region” or “CDR”, as defined below. The terms “hypervariable loop” and “complementarity determining region” are not strictly synonymous, since the hypervariable loops (HVs) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1983) and the limits of the HVs and the CDRs may be different in some VH and VL domains.

The CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3) in the light chain variable domain, and residues 31-35 or 31-35b (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Thus, the HVs may be comprised within the corresponding CDRs and references herein to the “hypervariable loops” of VH and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called the framework region (FR), as defined below. The variable domains of native heavy and light chains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively), largely adopting a β-sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen-binding site of antibodies. Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt just a small repertoire of main-chain conformations, called “canonical structures”. These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main-chain conformations.

-   “CDR”—As used herein, the term “CDR” or “complementarity determining     region” means the non-contiguous antigen combining sites found     within the variable region of both heavy and light chain     polypeptides. These particular regions have been described by Kabat     et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al.,     Sequences of protein of immunological interest. (1991), and by     Chothia et al., J. Mol. Biol. 196:901-917 (1987) and by MacCallum et     al., J. Mol. Biol. 262:732-745 (1996) where the definitions include     overlapping or subsets of amino acid residues when compared against     each other. The amino acid residues which encompass the CDRs as     defined by each of the above cited references are set forth for     comparison. Preferably, the term “CDR” is a CDR as defined by Kabat     based on sequence comparisons.

TABLE 1 CDR Definitions CDR Definitions Kabat¹ Chothia² MacCallum³ V_(II) CDR1 31-35 26-32 30-35 V_(H) CDR2 50-65 53-55 47-58 V_(H) CDR3  95-102  96-101  93-101 V_(L) CDR1 24-34 26-32 30-36 V_(L) CDR2 50-56 50-52 46-55 V_(L) CDR3 89-97 91-96 89-96 ¹Residue numbering follows the nomenclature of Kabat et al., supra ²Residue numbering follows the nomenclature of Chothia et al., supra ³Residue numbering follows the nomenclature of MacCallum et al., supra

-   “Framework region”—The term “framework region” or “FR region” as     used herein, includes the amino acid residues that are part of the     variable region, but are not part of the CDRs (e.g., using the Kabat     definition of CDRs). Therefore, a variable region framework is     between about 100-120 amino acids in length but includes only those     amino acids outside of the CDRs. For the specific example of a heavy     chain variable domain and for the CDRs as defined by Kabat et al.,     framework region 1 corresponds to the domain of the variable region     encompassing amino acids 1-30; framework region 2 corresponds to the     domain of the variable region encompassing amino acids 36-49;     framework region 3 corresponds to the domain of the variable region     encompassing amino acids 66-94, and framework region 4 corresponds     to the domain of the variable region from amino acids 103 to the end     of the variable region. The framework regions for the light chain     are similarly separated by each of the light claim variable region     CDRs. Similarly, using the definition of CDRs by Chothia et al. or     McCallum et al. the framework region boundaries are separated by the     respective CDR termini as described above. In preferred embodiments     the CDRs are as defined by Kabat.

In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen binding site as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the heavy and light variable domains show less inter-molecular variability in amino acid sequence and are termed the framework regions. The framework regions largely adopt a β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, these framework regions act to form a scaffold that provides for positioning the six CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen binding site formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface promotes the non-covalent binding of the antibody to the immunoreactive antigen epitope. The position of CDRs can be readily identified by one of ordinary skill in the art.

-   “Hinge region”—As used herein, the term “hinge region” includes the     portion of a heavy chain molecule that joins the CH1 domain to the     CH2 domain. This hinge region comprises approximately 25 residues     and is flexible, thus allowing the two N-terminal antigen binding     regions to move independently. Hinge regions can be subdivided into     three distinct domains: upper, middle, and lower hinge domains (Roux     et al. J. Immunol. 1998 161:4083). CD70 antibodies comprising a     “fully human” hinge region may contain one of the hinge region     sequences shown in Table 2 below.

TABLE 2 human hinge sequences IgG Upper hinge Middle hinge Lower hinge IgG1 EPKSCDKTHT CPPCP APELLGGP (SEQ ID NO: 320) (SEQ ID NO: 321) (SEQ ID NO: 322) IgG3 ELKTPLGDTTHT CPRCP (EPKSCDTPPPCPRCP)₃ APELLGGP (SEQ ID NO: 323) (SEQ ID NO: 324) (SEQ ID NO: 325) IgG4 ESKYGPP CPSCP APEFLGGP (SEQ ID NO: 326) (SEQ ID NO: 327) (SEQ ID NO: 328) IgG42 ERK CCVECPPPCP APPVAGP (SEQ ID NO: 329) (SEQ ID NO: 330) (SEQ ID NO: 331)

-   “CH2 domain”—As used herein the term “CH2 domain” includes the     portion of a heavy chain molecule that extends, e.g., from about     residue 244 to residue 360 of an antibody using conventional     numbering schemes (residues 244 to 360, Kabat numbering system; and     residues 231-340, EU numbering system, Kabat E A et al. Sequences of     Proteins of Immunological Interest. Bethesda, US Department of     Health and Human Services, NIH. 1991). The CH2 domain is unique in     that it is not closely paired with another domain. Rather, two     N-linked branched carbohydrate chains are interposed between the two     CH2 domains of an intact native IgG molecule. It is also well     documented that the CH3 domain extends from the CH2 domain to the     C-terminal of the IgG molecule and comprises approximately 108     residues. -   “Fragment”—The term “fragment” refers to a part or portion of an     antibody or antibody chain comprising fewer amino acid residues than     an intact or complete antibody or antibody chain. The term     “antigen-binding fragment” refers to a polypeptide fragment of an     immunoglobulin or antibody that binds antigen or competes with     intact antibody (i.e., with the intact antibody from which they were     derived) for antigen binding (i.e., specific binding to human CD70).     As used herein, the term “fragment” of an antibody molecule includes     antigen-binding fragments of antibodies, for example, an antibody     light chain variable domain (VL), an antibody heavy chain variable     domain (VH), a single chain antibody (scFv), a F(ab′)2 fragment, a     Fab fragment, an Fd fragment, an Fv fragment, and a single domain     antibody fragment (DAb). Fragments can be obtained, e.g., via     chemical or enzymatic treatment of an intact or complete antibody or     antibody chain or by recombinant means. -   “Valency”—As used herein the term “valency” refers to the number of     potential target binding sites in a polypeptide. Each target binding     site specifically binds one target molecule or specific site on a     target molecule. When a polypeptide comprises more than one target     binding site, each target binding site may specifically bind the     same or different molecules (e.g., may bind to different ligands or     different antigens, or different epitopes on the same antigen). The     subject binding molecules have at least one binding site specific     for a human CD70 molecule. -   “Specificity”—The term “specificity” refers to the ability to bind     (e.g., immunoreact with) a given target, e.g., CD70. A polypeptide     may be monospecific and contain one or more binding sites which     specifically bind a target or a polypeptide may be multispecific and     contain two or more binding sites which specifically bind the same     or different targets. In one embodiment, an antibody of the     invention is specific for more than one target. For example, in one     embodiment, a multispecific binding molecule of the invention binds     to CD70 and a second molecule expressed on a tumor cell. Exemplary     antibodies which comprise antigen binding sites that bind to     antigens expressed on tumor cells are known in the art and one or     more CDRs from such antibodies can be included in an antibody of the     invention. -   “Synthetic”—As used herein the term “synthetic” with respect to     polypeptides includes polypeptides which comprise an amino acid     sequence that is not naturally occurring. For example, non-naturally     occurring polypeptides which are modified forms of naturally     occurring polypeptides (e.g., comprising a mutation such as an     addition, substitution or deletion) or which comprise a first amino     acid sequence (which may or may not be naturally occurring) that is     linked in a linear sequence of amino acids to a second amino acid     sequence (which may or may not be naturally occurring) to which it     is not naturally linked in nature. -   “Engineered”—As used herein the term “engineered” includes     manipulation of nucleic acid or polypeptide molecules by synthetic     means (e.g. by recombinant techniques, in vitro peptide synthesis,     by enzymatic or chemical coupling of peptides or some combination of     these techniques). Preferably, the antibodies of the invention are     engineered, including for example, humanized and/or chimeric     antibodies, and antibodies which have been engineered to improve one     or more properties, such as antigen binding, stability/half-life or     effector function. -   “Modified antibody”—As used herein, the term “modified antibody”     includes synthetic forms of antibodies which are altered such that     they are not naturally occurring, e.g., antibodies that comprise at     least two heavy chain portions but not two complete heavy chains     (such as, domain deleted antibodies or minibodies); multispecific     forms of antibodies (e.g., bispecific, trispecific, etc.) altered to     bind to two or more different antigens or to different epitopes on a     single antigen); heavy chain molecules joined to scFv molecules and     the like. ScFv molecules are known in the art and are described,     e.g., in U.S. Pat. No. 5,892,019. In addition, the term “modified     antibody” includes multivalent forms of antibodies (e.g., trivalent,     tetravalent, etc., antibodies that bind to three or more copies of     the same antigen). In another embodiment, a modified antibody of the     invention is a fusion protein comprising at least one heavy chain     portion lacking a CH2 domain and comprising a binding domain of a     polypeptide comprising the binding portion of one member of a     receptor ligand pair.

The term “modified antibody” may also be used herein to refer to amino acid sequence variants of a CD70 antibody. It will be understood by one of ordinary skill in the art that a CD70 antibody may be modified to produce a variant CD70 antibody which varies in amino acid sequence in comparison to the CD70 antibody from which it was derived. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues may be made (e.g., in CDR and/or framework residues). Amino acid substitutions can include replacement of one or more amino acids with a naturally occurring or non-natural amino acid.

-   “Humanising substitutions”—As used herein, the term “humanising     substitutions” refers to amino acid substitutions in which the amino     acid residue present at a particular position in the VH or VL domain     of a CD70 antibody (for example a camelid-derived CD70 antibody) is     replaced with an amino acid residue which occurs at an equivalent     position in a reference human VH or VL domain. The reference human     VH or VL domain may be a VH or VL domain encoded by the human     germline. Humanising substitutions may be made in the framework     regions and/or the CDRs of a CD70 antibody, defined herein. -   “Humanised variants”—As used herein the term “humanised variant”     refers to a variant antibody which contains one or more “humanising     substitutions” compared to a reference CD70 antibody, wherein a     portion of the reference antibody (e.g. the VH domain and/or the VL     domain or parts thereof containing at least one CDR) has an amino     derived from a non-human species, and the “humanising substitutions”     occur within the amino acid sequence derived from a non-human     species. -   “Germlined variants”—The term “germlined variant” is used herein to     refer specifically to “humanised variants” in which the “humanising     substitutions” result in replacement of one or more amino acid     residues present at a particular position (s) in the VH or VL domain     of a CD70 antibody (for example a camelid-derived CD70 antibody)     with an amino acid residue which occurs at an equivalent position in     a reference human VH or VL domain encoded by the human germline. It     is typical that for any given “germlined variant”, the replacement     amino acid residues substituted into the germlined variant are taken     exclusively, or predominantly, from a single human germline-encoded     VH or VL domain. The terms “humanised variant” and “germlined     variant” are often used interchangeably herein. Introduction of one     or more “humanising substitutions” into a camelid-derived (e.g.     llama derived) VH or VL domain results in production of a “humanised     variant” of the camelid (llama)-derived VH or VL domain. If the     amino acid residues substituted in are derived predominantly or     exclusively from a single human germline-encoded VH or VL domain     sequence, then the result may be a “human germlined variant” of the     camelid (llama)-derived VH or VL domain. -   “Affinity variants”—As used herein, the term “affinity variant”     refers to a variant antibody which exhibits one or more changes in     amino acid sequence compared to a reference CD70 antibody, wherein     the affinity variant exhibits an altered affinity for the human CD70     protein in comparison to the reference antibody. Typically, affinity     variants will exhibit a changed affinity for human CD70, as compared     to the reference CD70 antibody. Preferably the affinity variant will     exhibit improved affinity for human CD70, as compared to the     reference CD70 antibody. The improvement may be apparent as a lower     K_(D), for human CD70, or a slower off-rate for human CD70 or an     alteration in the pattern of cross-reactivity with non-human CD70     homologues. Affinity variants typically exhibit one or more changes     in amino acid sequence in the CDRs, as compared to the reference     CD70 antibody. Such substitutions may result in replacement of the     original amino acid present at a given position in the CDRs with a     different amino acid residue, which may be a naturally occurring     amino acid residue or a non-naturally occurring amino acid residue.     The amino acid substitutions may be conservative or     non-conservative. -   “High human homology”—An antibody comprising a heavy chain variable     domain (VH) and a light chain variable domain (VL) will be     considered as having high human homology if the VH domains and the     VL domains, taken together, exhibit at least 90% amino acid sequence     identity to the closest matching human germline VH and VL sequences.     Antibodies having high human homology may include antibodies     comprising VH and VL domains of native non-human antibodies which     exhibit sufficiently high % sequence identity to human germline     sequences, including for example antibodies comprising VH and VL     domains of camelid conventional antibodies, as well as engineered,     especially humanised or germlined, variants of such antibodies and     also “fully human” antibodies.

In one embodiment the VH domain of the antibody with high human homology may exhibit an amino acid sequence identity or sequence homology of 80% or greater with one or more human VH domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VH domain of the polypeptide of the invention and the closest matching human germline VH domain sequence may be 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.

In one embodiment the VH domain of the antibody with high human homology may contain one or more (e.g. 1 to 10) amino acid sequence mis-matches across the framework regions FR1, FR2, FR3 and FR4, in comparison to the closest matched human VH sequence.

In another embodiment the VL domain of the antibody with high human homology may exhibit a sequence identity or sequence homology of 80% or greater with one or more human VL domains across the framework regions FR1, FR2, FR3 and FR4. In other embodiments the amino acid sequence identity or sequence homology between the VL domain of the polypeptide of the invention and the closest matching human germline VL domain sequence may be 85% or greater 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100%.

In one embodiment the VL domain of the antibody with high human homology may contain one or more (e.g. 1 to 10) amino acid sequence mis-matches across the framework regions FR1, FR2, FR3 and FR4, in comparison to the closest matched human VL sequence.

Before analyzing the percentage sequence identity between the antibody with high human homology and human germline VH and VL, the canonical folds may be determined, which allows the identification of the family of human germline segments with the identical combination of canonical folds for H1 and H2 or L1 and L2 (and L3). Subsequently the human germline family member that has the highest degree of sequence homology with the variable region of the antibody of interest is chosen for scoring the sequence homology. The determination of Chothia canonical classes of hypervariable loops L1, L2, L3, H1 and H2 can be performed with the bioinformatics tools publicly available on webpage www.bioinf.org.uk/abs/chothia.html.page. The output of the program shows the key residue requirements in a datafile. In these datafiles, the key residue positions are shown with the allowed amino acids at each position. The sequence of the variable region of the antibody of interest is given as input and is first aligned with a consensus antibody sequence to assign the Kabat numbering scheme. The analysis of the canonical folds uses a set of key residue templates derived by an automated method developed by Martin and Thornton (Martin et al., J. Mol. Biol. 263:800-815 (1996)).

With the particular human germline V segment known, which uses the same combination of canonical folds for H1 and H2 or L1 and L2 (and L3), the best matching family member in terms of sequence homology can be determined. With bioinformatics tools the percentage sequence identity between the VH and VL domain framework amino acid sequences of the antibody of interest and corresponding sequences encoded by the human germline can be determined, but actually manual alignment of the sequences can be applied as well. Human immunoglobulin sequences can be identified from several protein data bases, such as VBase (http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database (http://www.bioc.unizh.ch/antibody/Sequences/Germlines. To compare the human sequences to the V regions of VH or VL domains in an antibody of interest a sequence alignment algorithm such as available via websites like www.expasy.ch/tools/#align can be used, but also manual alignment with the limited set of sequences can be performed. Human germline light and heavy chain sequences of the families with the same combinations of canonical folds and with the highest degree of homology with the framework regions 1, 2, and 3 of each chain are selected and compared with the variable region of interest; also the FR4 is checked against the human germline JH and JK or JL regions.

Note that in the calculation of overall percent sequence homology the residues of FR1, FR2 and FR3 are evaluated using the closest match sequence from the human germline family with the identical combination of canonical folds. Only residues different from the closest match or other members of the same family with the same combination of canonical folds are scored (NB—excluding any primer-encoded differences). However, for the purposes of humanization, residues in framework regions identical to members of other human germline families, which do not have the same combination of canonical folds, can be considered “human”, despite the fact that these are scored “negative” according to the stringent conditions described above. This assumption is based on the “mix and match” approach for humanization, in which each of FR1, FR2, FR3 and FR4 is separately compared to its closest matching human germline sequence and the humanized molecule therefore contains a combination of different FRs as was done by Qu and colleagues (Qu et la., Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Ono et al., Mol. Immunol. 36:387-395 (1999)). The boundaries of the individual framework regions may be assigned using the IMGT numbering scheme, which is an adaptation of the numbering scheme of Chothia (Lefranc et al., NAR 27: 209-212 (1999); imgt.cines.fr).

Antibodies with high human homology may comprise hypervariable loops or CDRs having human or human-like canonical folds, as discussed in detail below. In one embodiment at least one hypervariable loop or CDR in either the VH domain or the VL domain of the antibody with high human homology may be obtained or derived from a VH or VL domain of a non-human antibody, for example a conventional antibody from a species of Camelidae, yet exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.

It is well established in the art that although the primary amino acid sequences of hypervariable loops present in both VH domains and VL domains encoded by the human germline are, by definition, highly variable, all hypervariable loops, except CDR H3 of the VH domain, adopt only a few distinct structural conformations, termed canonical folds (Chothia et al., J. Mol. Biol. 196:901-917 (1987); Tramontano et al. Proteins 6:382-94 (1989)), which depend on both the length of the hypervariable loop and presence of the so-called canonical amino acid residues (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Actual canonical structures of the hypervariable loops in intact VH or VL domains can be determined by structural analysis (e.g. X-ray crystallography), but it is also possible to predict canonical structure on the basis of key amino acid residues which are characteristic of a particular structure (discussed further below). In essence, the specific pattern of residues that determines each canonical structure forms a “signature” which enables the canonical structure to be recognised in hypervariable loops of a VH or VL domain of unknown structure; canonical structures can therefore be predicted on the basis of primary amino acid sequence alone.

The predicted canonical fold structures for the hypervariable loops of any given VH or VL sequence in an antibody with high human homology can be analysed using algorithms which are publicly available from www.bioinf.org.uk/abs/chothia.html, www.biochem.ucl.ac.uk/˜martin/antibodies.html and www.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html. These tools permit query VH or VL sequences to be aligned against human VH or VL domain sequences of known canonical structure, and a prediction of canonical structure made for the hypervariable loops of the query sequence.

In the case of the VH domain, H1 and H2 loops may be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled:

-   1. An identical length, determined by the number of residues, to the     closest matching human canonical structural class. -   2. At least 33% identity, preferably at least 50% identity with the     key amino acid residues described for the corresponding human H1 and     H2 canonical structural classes.     (note for the purposes of the foregoing analysis the H1 and H2 loops     are treated separately and each compared against its closest     matching human canonical structural class)

The foregoing analysis relies on prediction of the canonical structure of the H1 and H2 loops of the antibody of interest. If the actual structures of the H1 and H2 loops in the antibody of interest are known, for example based on X-ray crystallography, then the H1 and H2 loops in the antibody of interest may also be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by ±1 or ±2 amino acids) but the actual structure of the H1 and H2 loops in the antibody of interest matches the structure of a human canonical fold.

Key amino acid residues found in the human canonical structural classes for the first and second hypervariable loops of human VH domains (H1 and H2) are described by Chothia et al., J. Mol. Biol. 227:799-817 (1992), the contents of which are incorporated herein in their entirety by reference. In particular, Table 3 on page 802 of Chothia et al., which is specifically incorporated herein by reference, lists preferred amino acid residues at key sites for H1 canonical structures found in the human germline, whereas Table 4 on page 803, also specifically incorporated by reference, lists preferred amino acid residues at key sites for CDR H2 canonical structures found in the human germline.

In one embodiment, both H1 and H2 in the VH domain of the antibody with high human homology exhibit a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.

Antibodies with high human homology may comprise a VH domain in which the hypervariable loops H1 and H2 form a combination of canonical fold structures which is identical to a combination of canonical structures known to occur in at least one human germline VH domain. It has been observed that only certain combinations of canonical fold structures at H1 and H2 actually occur in VH domains encoded by the human germline. In an embodiment H1 and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, yet form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline or somatically mutated VH domain. In non-limiting embodiments H1 and H2 in the VH domain of the antibody with high human homology may be obtained from a VH domain of a non-human species, e.g. a Camelidae species, and form one of the following canonical fold combinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5.

An antibody with high human homology may contain a VH domain which exhibits both high sequence identity/sequence homology with human VH, and which contains hypervariable loops exhibiting structural homology with human VH.

It may be advantageous for the canonical folds present at H1 and H2 in the VH domain of the antibody with high human homology, and the combination thereof, to be “correct” for the human VH germline sequence which represents the closest match with the VH domain of the antibody with high human homology in terms of overall primary amino acid sequence identity. By way of example, if the closest sequence match is with a human germline VH3 domain, then it may be advantageous for H1 and H2 to form a combination of canonical folds which also occurs naturally in a human VH3 domain. This may be particularly important in the case of antibodies with high human homology which are derived from non-human species, e.g. antibodies containing VH and VL domains which are derived from camelid conventional antibodies, especially antibodies containing humanised camelid VH and VL domains.

Thus, in one embodiment the VH domain of the CD70 antibody with high human homology may exhibit a sequence identity or sequence homology of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VH domain across the framework regions FR1, FR2, FR3 and FR4, and in addition H1 and H2 in the same antibody are obtained from a non-human VH domain (e.g. derived from a Camelidae species), but form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VH domain.

In other embodiments, L1 and L2 in the VL domain of the antibody with high human homology are each obtained from a VL domain of a non-human species (e.g. a camelid-derived VL domain), and each exhibits a predicted or actual canonical fold structure which is substantially identical to a canonical fold structure which occurs in human antibodies.

As with the VH domains, the hypervariable loops of VL domains of both VLambda and VKappa types can adopt a limited number of conformations or canonical structures, determined in part by length and also by the presence of key amino acid residues at certain canonical positions.

Within an antibody of interest having high human homology, L1, L2 and L3 loops obtained from a VL domain of a non-human species, e.g. a Camelidae species, may be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if at least the first, and preferable both, of the following criteria are fulfilled:

-   1. An identical length, determined by the number of residues, to the     closest matching human structural class. -   2. At least 33% identity, preferably at least 50% identity with the     key amino acid residues described for the corresponding human L1 or     L2 canonical structural classes, from either the VLambda or the     VKappa repertoire.     (note for the purposes of the foregoing analysis the L1 and L2 loops     are treated separately and each compared against its closest     matching human canonical structural class)

The foregoing analysis relies on prediction of the canonical structure of the L1, L2 and L3 loops in the VL domain of the antibody of interest. If the actual structure of the L1, L2 and L3 loops is known, for example based on X-ray crystallography, then L1, L2 or L3 loops derived from the antibody of interest may also be scored as having a canonical fold structure “substantially identical” to a canonical fold structure known to occur in human antibodies if the length of the loop differs from that of the closest matching human canonical structural class (typically by ±1 or ±2 amino acids) but the actual structure of the Camelidae loops matches a human canonical fold.

Key amino acid residues found in the human canonical structural classes for the CDRs of human VLambda and VKappa domains are described by Morea et al. Methods, 20: 267-279 (2000) and Martin et al., J. Mol. Biol., 263:800-815 (1996). The structural repertoire of the human VKappa domain is also described by Tomlinson et al. EMBO J. 14:4628-4638 (1995), and that of the VLambda domain by Williams et al. J. Mol. Biol., 264:220-232 (1996). The contents of all these documents are to be incorporated herein by reference.

L1 and L2 in the VL domain of an antibody with high human homology may form a combination of predicted or actual canonical fold structures which is identical to a combination of canonical fold structures known to occur in a human germline VL domain. In non-limiting embodiments L1 and L2 in the VLambda domain of an antibody with high human homology (e.g. an antibody containing a camelid-derived VL domain or a humanised variant thereof) may form one of the following canonical fold combinations: 11-7, 13-7(A,B,C), 14-7(A,B), 12-11, 14-11 and 12-12 (as defined in Williams et al. J. Mol. Biol. 264:220-32 (1996) and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVL.html). In non-limiting embodiments L1 and L2 in the Vkappa domain may form one of the following canonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (as defined in Tomlinson et al. EMBO J. 14:4628-38 (1995) and as shown on http://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK.html).

In a further embodiment, all three of L1, L2 and L3 in the VL domain of an antibody with high human homology may exhibit a substantially human structure. It is preferred that the VL domain of the antibody with high human homology exhibits both high sequence identity/sequence homology with human VL, and also that the hypervariable loops in the VL domain exhibit structural homology with human VL.

In one embodiment, the VL domain of a CD70 antibody with high human homology may exhibit a sequence identity of 80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% or greater, or up to 99% or even 100% with a human VL domain across the framework regions FR1, FR2, FR3 and FR4, and in addition hypervariable loop L1 and hypervariable loop L2 may form a combination of predicted or actual canonical fold structures which is the same as a canonical fold combination known to occur naturally in the same human VL domain.

It is, of course, envisaged that VH domains exhibiting high sequence identity/sequence homology with human VH, and also structural homology with hypervariable loops of human VH will be combined with VL domains exhibiting high sequence identity/sequence homology with human VL, and also structural homology with hypervariable loops of human VL to provide antibodies with high human homology containing VH/VL pairings (e.g camelid-derived VH/V1 pairings) with maximal sequence and structural homology to human-encoded VH/VL pairings.

As summarised above, the invention relates, at least in part, to antibodies, and antigen binding fragments thereof, that bind to human CD70 with high affinity. The properties and characteristics of the CD70 antibodies, and antibody fragments, according to the invention will now be described in further detail.

CD70 Binding and Affinity

In certain aspects, the antibodies, and antigen binding fragments thereof, provided herein bind to human CD70 with high affinity. Antibodies, or antigen binding fragments thereof, which bind to human CD70 with high affinity may exhibit a binding affinity (K_(D)) for human CD70, and more particularly the extracellular domain of human CD70, of about 10 nM or less, or 1 nM or less, or 0.1 nM or less, or 10 pM or less.

The CD70 antibody (or antigen binding fragment) may comprise VH and VL domains which, when the VH and VL domains are expressed in the form of a Fab, exhibit a dissociation off-rate for human CD70 binding of less than 7×10⁻⁴ s⁻¹, preferably less than 5×10⁻⁴ s⁻¹, or less than 2×10⁻⁴ s⁻¹, less than 1×10⁻⁴ s⁻¹. Typically the off rate will fall in the range of from 0.4×10⁻⁴ s⁻¹ to 4.8×10⁻⁴ s⁻¹. Binding affinity (K_(D)) and dissociation rate (K_(off)) can be measured using standard techniques well known to persons skilled in the art, such as for example surface plasmon resonance (BIAcore), as described in the accompanying examples. In brief, 50 μl of sample to be tested is added to 200 μl PBS+0.02% Tween, and diluted 1/400 as follows: 5 μl of sample (1 mg/ml)+195 μl HBS-EP+(Biacore buffer), further diluted 10 μl+90 μl HVS-EP+=2.5 μg/ml. Biacore analysis is performed at room temperature using a highly CD70 coated CM5 chip (4000 RU) using the manufacturer's supplied protocol.

In this regard, it should be noted that although the off-rate is measured for VH and VL combinations in the form of Fabs, this does not mean that the VH and VL domains contribute equally to CD70 binding. For many VH/VL combinations, it is the VH domain which mainly contributes to CD70 binding, with the VL domain contributing to solubility and/or stability of the VH/VL pairing.

Binding of the CD70 antibodies described herein, or Fab fragments thereof, to recombinant human CD70 may also be assessed by ELISA.

The CD70 antibodies described herein may further exhibit binding to CD70 expressed on the surface of intact cells, e.g. 786-O renal carcinoma cells and other cancer cell lines. Binding of CD70 antibodies to CD70-expressing cells may be assessed by flow cytometry. The results presented in the accompanying examples demonstrate that preferred CD70 antibodies, including variant 41D12 (when expressed as non-fucosylated IgG1 ARGX-110), exhibit very strong binding to cell-surface CD70. Indeed, for many cell lines ARGX-110 exhibits stronger binding than comparator prior art antibody SGN70.

It is particularly noteworthy that ARGX-110 exhibits strong binding to cell lines which express CD70 at low copy number, including inter alia the cell lines Raji, SU-DHL-6, MHHPREB1, Mino, Mec1, JVM-2, HH and EBC-1, and also to cancer cells isolated from patients (e.g. CLL patients), in both cases the binding of ARGX-110 is markedly stronger than SGN70. The “improved” binding to low copy number cell lines and CLL patient materials may in large part reflect the extremely high affinity of ARGX-110 for recombinant CD70. These binding characteristics are supportive of a particular utility of ARGX-110 (based on 41D12) and other CD70 antibodies described herein with similar properties in cancer treatment, particularly when utilised as an IgG exhibiting potent effector function (e.g. non-fucosylated IgG1) rather than as an immunoconjugate linked to a cytotoxic or cytostatic moiety. The significance of high affinity binding (i.e. higher affinity than can be achieved with prior art CD70 antibodies) to recombinant CD70 and cell surface CD70 with regard to clinical utility of the antibodies is demonstrated in the accompanying examples by experiments in which PBMC samples are “spiked” with cancer cell lines and then treated with CD70 antibodies. In this system treatment with ARGX-110 produced significantly more lysis of the target cancer cells than the comparator CD70 antibodies MDX1411 and SGN70.

The CD70 antibodies described herein exhibit high affinity binding to human CD70, and more specifically the extracellular domain of human CD70, but cross-reactivity with non-human homologues of CD70 is not excluded. Indeed, it is an advantageous feature of many of the CD70 antibodies described herein, including 27B3 and its germlined variants, particularly 41D12, that in addition to high affinity binding to human CD70 they also cross-react with simian CD70 homologs, specifically the CD70 homologs of rhesus macaque and cynomolgus monkey.

In this regard, a CD70 antibody can be considered to exhibit cross-reactivity with a simian CD70 homolog, for example the CD70 homologs of rhesus macaque and cynomolgus monkey, if the difference in IC50 for human versus simian (rhesus or cynomolgus monkey) CD70 in a CD70-CD27 inhibition ELISA, such as that described in the accompanying example 20.2, is less than 5-fold, preferably less than 3-fold or less than 2-fold. The binding affinity, and therefore blocking potency, for human and simian CD70 should be broadly comparable.

Interaction Between CD70 and CD27

In certain aspects, the CD70 antibodies, or antigen binding fragments thereof, provided herein may bind to human CD70 with high affinity and block the interaction between CD70 and CD27.

The ability of CD70 antibodies, or antigen binding fragments thereof, to block binding of CD70 to CD27 may be assessed by Elisa using either captured recombinant CD70 (Flag-TNC-CD70) or directed coated CD70 and recombinant CD27-Fc. The CD70 antibodies described herein may inhibit the interaction between CD70 and CD27 with an IC50 of 300 ng/ml or less, or 200 ng/ml or less, or 110 ng/ml or less, or 70 ng/ml or less, or 50 ng/ml or less.

The ability of CD70 antibodies, or antigen binding fragments thereof, to block binding of CD70 to CD27 may also be assessed in an assay based on co-culture of Raji cells (human B cell lymphoma) and HT1080-CD27 cells (human epithelial cell line transfected with CD27), as described in the accompanying examples. The CD70 antibodies described which inhibit the interaction between CD70 and CD27 may exhibit an IC50 in this co-culture assay of 500 ng/ml or less, or 300 ng/ml or less, or 100 ng/ml or less, or 50 ng/ml or less, or 30 ng/ml or less.

The preferred CD70 antibodies based on 27B3 and germlined variants thereof, including 41D12 (ARGX-110) exhibit potent blocking of the CD70/CD27 interaction in the Elisa system. Indeed, ARGX-110 is significantly more potent in blocking CD70/CD27 interactions than comparator antibodies SGN70 and MDX1411. The interaction between CD70 and CD27 may contribute to tumour cell survival, proliferation and/or immune suppression within the tumour microenvironment. Accordingly, potent inhibition of the CD70/CD27 interaction, in addition to high affinity binding to CD70, may contribute to improved clinical outcome in the treatment of certain CD70-expressing cancers or immunological disorders, for example autoimmune diseases and cancers which co-express CD70 and CD27.

In other embodiments, the CD70 antibodies, or antigen binding fragments thereof, may bind to human CD70 but not inhibit the interaction between CD70 and CD27. CD70 antibodies which bind CD70 but do not inhibit the interaction between CD70 and CD27 can be assessed based on Elisa using either captured recombinant CD70 (Flag-TNC-CD70) or directed coated CD70 and recombinant CD27-Fc. As demonstrated in the accompanying examples, a number of Fabs have been identified which bind CD70 but do not inhibit the CD70/CD27 interaction to a significant extent. In particular, the Fab clone identified herein as 59D10 exhibits strong binding to recombinant CD70 as measured by Biacore but does not block the CD70/CD27 interaction to a significant extent, when assessed by ELISA. CD70 antibodies described herein which bind CD70 but do not inhibit the interaction between CD70 and CD27 may still possess intact antibody effector functions, i.e. one or more of ADCC, CDC, ADCP or ADC and inhibit tumour cell growth in vivo. CD70 antibodies which bind CD70 with high affinity but are non-blocking may be advantageously utilised as “one-armed” antibodies, or PEGylated Fab products or in any other antibody format which provides a strict monovalent interaction with a target cell expressing CD70.

CD70 Epitopes

In certain aspects, the CD70 antibodies described herein bind to epitopes within the extracellular domain of human CD70.

The term “epitope” refers to the portion(s) of an antigen (e.g. human CD70) that contact an antibody. Epitopes can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.

The CD70 antibodies provided herein may bind to different (overlapping or non-overlapping) epitopes within the extracellular domain of the human CD70 protein. For example, the Fabs denoted 1C2 and 9E1 in the accompanying examples clearly bind to different, non-overlapping epitopes on human CD70.

As noted elsewhere herein, the preferred CD70 antibody 41D12 (ARGX-110) exhibits a particularly useful combination of binding characteristics which is not exhibited by any known prior art CD70 antibody, namely:

-   -   (a) binding within the amino acid sequence HIQVTLAICSS (SEQ ID         NO:342) in human CD70;     -   (b) cross-reactivity with CD70 homologs of rhesus macaque         (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus);     -   (c) binding to native human CD70 and heat denatured recombinant         human CD70.

For any given CD70 antibody, the ability to bind human CD70 within the amino acid sequence HIQVTLAICSS (SEQ ID NO:342) can be readily determined by a person of ordinary skill in the art, for example using the mouse-human chimeric CD70 binding analysis described in the accompanying example 20.4.

Cross-reactivity with simian CD70 homologs can also be readily determined by a person of ordinary skill in the art, for example by FACs analysis, surface plasmon resonance (Biacore™) or using a CD70-CD27 inhibition ELISA. In this regard, a CD70 antibody can be considered to exhibit cross-reactivity with a simian CD70 homolog, for example the CD70 homologs of rhesus macaque and cynomolgus monkey, if the difference in IC50 for human versus simian (rhesus or cynomolgus monkey) CD70 in a CD70-CD27 inhibition ELISA, such as that described in the accompanying example 20.2, is less than 5-fold, preferably less than 3-fold or less than 2-fold.

The CD70 antibodies described herein may also demonstrate the ability to hind both native human CD70 and heat denatured recombinant human CD70. In this connection, binding to “native” human CD70 is indicated by binding to CD70 expressed on the surface of a CD70-expressing cell, such as any of the CD70+ cell lines described in the accompanying examples, or even natural CD70-expressing cells isolated from patient material (e.g. cells isolated from CLL patients as in the accompanying examples), activated T cells, etc. Binding to “native” human CD70 can thus be easily tested by a person of ordinary skill in the art.

Binding to heat denatured recombinant human CD70 can be tested as described in the accompanying example 20.3. It is preferred that the CD70 antibody should exhibit an OD at 620 nm of 0.6 or greater in this assay, in order to demonstrate significant binding to heat denatured recombinant human CD70.

Partial or Slow Internalisation

The CD70 antibodies described herein, including the preferred CD70 antibody 41D12 (ARGX-110), exhibit partial internalisation in renal carcinoma cell lines, meaning that when antibody internalisation is tested in 786-O renal carcinoma cells a substantial proportion of the bound antibody, i.e. at least 30-40%, remains external after 6 hours, and even after 24 hours, incubation at 37° C. In addition, the CD70 antibodies may also exhibit a slow rate of internalisation. In this regard, a CD70 antibody is considered to exhibit slow internalisation if it is internalised at a slower rate than a reference CD70 antibody 9D1 (VH SEQ ID NO:178; VL SEQ ID NO:190).

As demonstrated in the accompanying examples, different cancer cell lines exhibit significant differences in the degree of internalisation of CD70 antibodies. It is particularly significant that the majority of cancer cell lines exhibit less than 30%, and even less than 10%, internalisation of bound CD70 antibody, even after 6 hours incubation with ARGX-110. These results are clearly supportive of the utility of CD70 antibodies with potent effector function (including variants engineered for enhanced effector function) in the treatment of CD70-expressing cancers. It has previously been reported in the scientific literature that CD70 is an “internalising” target; hence it has been proposed to develop CD70 antibody-drug immunoconjugates for treatment of both CD70-expressing cancers and immunological diseases. Therefore, the results presented herein, which conclusively demonstrate that internalisation of cell-surface bound CD70 antibodies is a rare event and that the majority of CD70-expressing cell lines do not internalise bound CD70 antibody to a significant extent, are extremely surprising.

Camelid-derived CD70 Antibodies

In yet other aspects, the antibodies or antigen binding fragments thereof described herein may comprise at least one hypervariable loop or complementarity determining region obtained from a VH domain or a VL domain of a species in the family Camelidae. In particular, the antibody or antigen binding fragment may comprise VH and/or VL domains, or CDRs thereof, obtained by active immunisation of outbred camelids, e.g. llamas, with a human CD70 antigen.

By “hypervariable loop or complementarity determining region obtained from a VH domain or a VL domain of a species in the family Camelidae” is meant that that hypervariable loop (HV) or CDR has an amino acid sequence which is identical, or substantially identical, to the amino acid sequence of a hypervariable loop or CDR which is encoded by a Camelidae immunoglobulin gene. In this context “immunoglobulin gene” includes germline genes, immunoglobulin genes which have undergone rearrangement, and also somatically mutated genes. Thus, the amino acid sequence of the HV or CDR obtained from a VH or VL domain of a Camelidae species may be identical to the amino acid sequence of a HV or CDR present in a mature Camelidae conventional antibody. The term “obtained from” in this context implies a structural relationship, in the sense that the HVs or CDRs of the CD70 antibody embody an amino acid sequence (or minor variants thereof) which was originally encoded by a Camelidae immunoglobulin gene. However, this does not necessarily imply a particular relationship in terms of the production process used to prepare the CD70 antibody.

Camelid-derived CD70 antibodies may be derived from any camelid species, including inter alia, llama, dromedary, alpaca, vicuna, guanaco or camel.

CD70 antibodies comprising camelid-derived VH and VL domains, or CDRs thereof, are typically recombinantly expressed polypeptides, and may be chimeric polypeptides. The term “chimeric polypeptide” refers to an artificial (non-naturally occurring) polypeptide which is created by juxtaposition of two or more peptide fragments which do not otherwise occur contiguously. Included within this definition are “species” chimeric polypeptides created by juxtaposition of peptide fragments encoded by two or more species, e.g. camelid and human.

Camelid-derived CDRs may comprise one of the CDR sequences shown as SEQ ID Nos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 and 268 (light chain CDR1).

In one embodiment the entire VH domain and/or the entire VL domain may be obtained from a species in the family Camelidae. In specific embodiments, the camelid-derived VH domain may comprise the amino acid sequence shown as SEQ ID NOs: 177-188, 212-223, 274 or 275, whereas the camelid-derived VL domain may comprise the amino acid sequence show as SEQ ID Nos:189-211, 230-245, 276 or 277 (VL). The camelid-derived VH domain and/or the camelid-derived VL domain may then be subject to protein engineering, in which one or more amino acid substitutions, insertions or deletions are introduced into the camelid amino acid sequence. These engineered changes preferably include amino acid substitutions relative to the camelid sequence. Such changes include “humanisation” or “germlining” wherein one or more amino acid residues in a camelid-encoded VH or VL domain are replaced with equivalent residues from a homologous human-encoded VH or VL domain.

Isolated camelid VH and VL domains obtained by active immunisation of a camelid (e.g. llama) with a human CD70 antigen can be used as a basis for engineering antigen binding polypeptides according to the invention. Starting from intact camelid VH and VL domains, it is possible to engineer one or more amino acid substitutions, insertions or deletions which depart from the starting camelid sequence. In certain embodiments, such substitutions, insertions or deletions may be present in the framework regions of the VH domain and/or the VL domain. The purpose of such changes in primary amino acid sequence may be to reduce presumably unfavourable properties (e.g. immunogenicity in a human host (so-called humanization), sites of potential product heterogeneity and or instability (glycosylation, deamidation, isomerisation, etc.) or to enhance some other favourable property of the molecule (e.g. solubility, stability, bioavailability, etc.). In other embodiments, changes in primary amino acid sequence can be engineered in one or more of the hypervariable loops (or CDRs) of a Camelidae VH and/or VL domain obtained by active immunisation. Such changes may be introduced in order to enhance antigen binding affinity and/or specificity, or to reduce presumably unfavourable properties, e.g. immunogenicity in a human host (so-called humanization), sites of potential product heterogeneity and or instability, glycosylation, deamidation, isomerisation, etc., or to enhance some other favourable property of the molecule, e.g. solubility, stability, bioavailability, etc.

Thus, in one embodiment, the invention provides a variant CD70 antibody which contains at least one amino acid substitution in at least one framework or CDR region of either the VH domain or the VL domain in comparison to a camelid-derived VH or VL domain, examples of which include but are not limited to the camelid VH domains comprising the amino acid sequences shown as SEQ ID NO: 177-188, 212-223, 274 or 275, and the camelid VL domains comprising the amino acid sequences show as SEQ ID NO: 189-211, 230-245, 276 or 277.

In other embodiments, there are provided “chimeric” antibody molecules comprising camelid-derived VH and VL domains (or engineered variants thereof) and one or more constant domains from a non-camelid antibody, for example human-encoded constant domains (or engineered variants thereof). In such embodiments it is preferred that both the VH domain and the VL domain are obtained from the same species of camelid, for example both VH and VL may be from Lama glama or both VH and VL may be from Lama pacos (prior to introduction of engineered amino acid sequence variation). In such embodiments both the VH and the VL domain may be derived from a single animal, particularly a single animal which has been actively immunised with a human CD70 antigen.

As an alternative to engineering changes in the primary amino acid sequence of Camelidae VH and/or VL domains, individual camelid-derived hypervariable loops or CDRs, or combinations thereof, can be isolated from camelid VH/VL domains and transferred to an alternative (i.e. non-Camelidae) framework, e.g. a human VH/VL framework, by CDR grafting. In particular, non-limiting, embodiments the camelid-derived CDRs may be selected from CDRs having the amino acid sequences shown as SEQ ID NOs: 49-59 (heavy chain CDR3), or SEQ ID Nos: 26-37 (heavy chain CDR2) or SEQ ID Nos: 10-20 (heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs: 148-168 (light chain CDR3), or SEQ ID Nos: 109-128 (light chain CDR2) or SEQ ID Nos:77-95 (light chain CDR1).

CD70 antibodies comprising camelid-derived VH and VL domains, or CDRs thereof, can take various different embodiments in which both a VH domain and a VL domain are present. The term “antibody” herein is used in the broadest sense and encompasses, but is not limited to, monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), so long as they exhibit the appropriate immunological specificity for a human CD70 protein. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes) on the antigen, each monoclonal antibody is directed against a single determinant or epitope on the antigen.

“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable domain thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, bi-specific Fab′ s, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, a single chain variable fragment (scFv) and multispecific antibodies formed from antibody fragments (see Holliger and Hudson, Nature Biotechnol. 23:1126-36 (2005), the contents of which are incorporated herein by reference).

In non-limiting embodiments, CD70 antibodies comprising camelid-derived VH and VL domains, or CDRs thereof, may comprise CH1 domains and/or CL domains, the amino acid sequence of which is fully or substantially human. Where the antigen binding polypeptide of the invention is an antibody intended for human therapeutic use, it is typical for the entire constant region of the antibody, or at least a part thereof, to have fully or substantially human amino acid sequence. Therefore, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) may be fully or substantially human with respect to it's amino acid sequence.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) may all have fully or substantially human amino acid sequence. In the context of the constant region of a humanised or chimeric antibody, or an antibody fragment, the term “substantially human” refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% with a human constant region. The term “human amino acid sequence” in this context refers to an amino acid sequence which is encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes. The invention also contemplates polypeptides comprising constant domains of “human” sequence which have been altered, by one or more amino acid additions, deletions or substitutions with respect to the human sequence, excepting those embodiments where the presence of a “fully human” hinge region is expressly required.

The presence of a “fully human” hinge region in the CD70 antibodies of the invention may be beneficial both to minimise immunogenicity and to optimise stability of the antibody.

As discussed elsewhere herein, it is contemplated that one or more amino acid substitutions, insertions or deletions may be made within the constant region of the heavy and/or the light chain, particularly within the Fc region. Amino acid substitutions may result in replacement of the substituted amino acid with a different naturally occurring amino acid, or with a non-natural or modified amino acid. Other structural modifications are also permitted, such as for example changes in glycosylation pattern (e.g. by addition or deletion of N- or O-linked glycosylation sites). Depending on the intended use of the antibody, it may be desirable to modify the antibody of the invention with respect to its binding properties to Fc receptors, for example to modulate effector function. For example cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp. Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Alternatively, a CD70 antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). The invention also contemplates immunoconjugates comprising an antibody as described herein conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Fc regions may also be engineered for half-life extension, as described by Chan and Carter, Nature Reviews: Immunology, Vol. 10, pp 301-316, 2010, incorporated herein by reference.

In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids.

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for the CD70 target antigen. Such carbohydrate modifications can be accomplished by; for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen.

Also envisaged are variant CD70 antibodies having an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or a non-fucosylated antibody (as described by Natsume et al., Drug Design Development and Therapy, Vol. 3, pp 7-16, 2009) or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC activity of antibodies, producing typically 10-fold enhancement of ADCC relative to an equivalent antibody comprising a “native” human Fc domain. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation enzymatic machinery (as described by Yamane-Ohnuki and Satoh, mAbs 1:3, 230-236, 2009). Examples of non-fucosylated antibodies with enhanced ADCC function are those produced using the Potelligent™ technology of BioWa Inc.

The invention can, in certain embodiments, encompass chimeric Camelidae/human antibodies, and in particular chimeric antibodies in which the VH and VL domains are of fully camelid sequence (e.g. Llama or alpaca) and the remainder of the antibody is of fully human sequence. CD70 antibodies can include antibodies comprising “humanised” or “germlined” variants of camelid-derived VH and VL domains, or CDRs thereof, and camelid/human chimeric antibodies, in which the VH and VL domains contain one or more amino acid substitutions in the framework regions in comparison to camelid VH and VL domains obtained by active immunisation of a camelid with a human CD70 antigen. Such “humanisation” increases the % sequence identity with human germline VH or VL domains by replacing mis-matched amino acid residues in a starting Camelidae VH or VL domain with the equivalent residue found in a human germline-encoded VH or VL domain.

CD70 antibodies may also be CDR-grafted antibodies in which CDRs (or hypervariable loops) derived from a camelid antibody, for example an camelid CD70 antibody raised by active immunisation with human CD70 protein, or otherwise encoded by a camelid gene, are grafted onto a human VH and VL framework, with the remainder of the antibody also being of fully human origin. Such CDR-grafted CD70 antibodies may contain CDRs having the amino acid sequences shown as SEQ ID Nos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 and 268 (light chain CDR1).

Humanised, chimeric and CDR-grafted CD70 antibodies as described above, particularly antibodies comprising hypervariable loops or CDRs derived from active immunisation of camclids with a human CD70 antigen, can be readily produced using conventional recombinant DNA manipulation and expression techniques, making use of prokaryotic and eukaryotic host cells engineered to produce the polypeptide of interest and including but not limited to bacterial cells, yeast cells, mammalian cells, insect cells, plant cells, some of them as described herein and illustrated in the accompanying examples.

Camelid-derived CD70 antibodies include variants wherein the hypervariable loop(s) or CDR(s) of the VH domain and/or the VL domain are obtained from a conventional camelid antibody raised against human CD70, but wherein at least one of said (camelid-derived) hypervariable loops or CDRs has been engineered to include one or more amino acid substitutions, additions or deletions relative to the camelid-encoded sequence. Such changes include “humanisation” of the hypervariable loops/CDRs. Camelid-derived HVs/CDRs which have been engineered in this manner may still exhibit an amino acid sequence which is “substantially identical” to the amino acid sequence of a camelid-encoded HV/CDR. In this context, “substantial identity” may permit no more than one, or no more than two amino acid sequence mis-matches with the camelid-encoded HV/CDR. Particular embodiments of the CD70 antibody may contain humanised variants of the CDR sequences shown as SEQ ID Nos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 and 268 (light chain CDR1).

The camelid-derived CD70 antibodies provided herein may be of any isotype. Antibodies intended for human therapeutic use will typically be of the IgA, IgD, IgE IgG, IgM type, often of the IgG type, in which case they can belong to any of the four sub-classes IgG1, IgG2a and b, IgG3 or IgG4. Within each of these sub-classes it is permitted to make one or more amino acid substitutions, insertions or deletions within the Fc portion, or to make other structural modifications, for example to enhance or reduce Fc-dependent functionalities.

Humanisation of Camelid-derived VH and VL Domains

Camelid conventional antibodies provide an advantageous starting point for the preparation of antibodies with utility as human therapeutic agents due to the following factors, discussed in U.S. Ser. No. 12/497,239 which is incorporated herein by reference:

-   1) High % sequence homology between camelid VH and VL domains and     their human counterparts; -   2) High degree of structural homology between CDRs of camelid VH and     VL domains and their human counterparts (i.e. human-like canonical     fold structures and human-like combinations of canonical folds).

The camelid (e.g. llama) platform also provides a significant advantage in terms of the functional diversity of the CD70 antibodies which can be obtained.

The utility of CD70 antibodies comprising camelid VH and/or camelid VL domains for human therapy can be improved still further by “humanisation” of natural camelid VH and VL domains, for example to render them less immunogenic in a human host. The overall aim of humanisation is to produce a molecule in which the VH and VL domains exhibit minimal immunogenicity when introduced into a human subject, whilst retaining the specificity and affinity of the antigen binding site formed by the parental VH and VL domains.

One approach to humanisation, so-called “germlining”, involves engineering changes in the amino acid sequence of a camelid VH or VL domain to bring it closer to the germline sequence of a human VH or VL domain.

Determination of homology between a camelid VH (or VL) domain and human VH (or VL) domains is a critical step in the humanisation process, both for selection of camelid amino acid residues to be changed (in a given VH or VL domain) and for selecting the appropriate replacement amino acid residue(s).

An approach to humanisation of camelid conventional antibodies has been developed based on alignment of a large number of novel camelid VH (and VL) domain sequences, typically somatically mutated VH (or VL) domains which are known to bind a target antigen, with human germline VH (or VL) sequences, human VH (and VL) consensus sequences, as well as germline sequence information available for llama pacos. The following passages outline the principles which can be applied to (i) select “camelid” amino acid residues for replacement in a camelid-derived VH or VL domain or a CDR thereof, and (ii) select replacement “human” amino acid residues to substitute in, when humanising any given camelid VH (or VL) domain. This approach can be used to prepare humanised variants of camelid-derived CDRs having the amino acid sequences shown as SEQ ID Nos: 49-59, 262 or 263 (heavy chain CDR3), or SEQ ID Nos: 26-37, 249, 258 or 259 (heavy chain CDR2) or SEQ ID Nos: 10-20, 248, 256 or 257 (heavy chain CDR1) or one of the CDR sequences shown as SEQ ID NOs: 148-168, 271 or 273 (light chain CDR3), or SEQ ID Nos: 109-128 or 270 (light chain CDR2) or SEQ ID Nos:77-95, or 250-253, 267 or 268 (light chain CDR1), and also for humanisation of camelid-derived VH domains having the sequences shown as SEQ ID NOs: 177-188, 212-223, 274 or 275 and of camelid-derived VL domains having the sequences shown as SEQ ID Nos:189-211, 230-245, 276 or 277.

-   Step 1. Select human (germline) family and member of this family     that shows highest homology/identity to the mature camelid sequence     to be humanised. A general procedure for identifying the closest     matching human germline for any given camelid VH (or VL) domain is     outlined below. -   Step 2. Select specific human germline family member used to     germline against. Preferably this is the germline with the highest     homology or another germline family member from the same family. -   Step 3. Identify the preferred positions considered for germlining     on the basis of the table of amino acid utilisation for the camelid     germline that is closest to the selected human germline. -   Step 4. Try to change amino acids in the camelid germline that     deviate from the closest human germline; germlining of FR residues     is preferred over CDR residues. -   a. Preferred are positions that are deviating from the selected     human germline used to germline against, for which the amino acid     found in the camelid sequence does not match with the selected     germline and is not found in other germlines of the same subclass     (both for V as well as for J encoded FR amino acids). -   b. Positions that are deviating from the selected human germline     family member but which are used in other germlines of the same     family may also be addressed in the germlining process. -   c. Additional mismatches (e.g. due to additional somatic mutations)     towards the selected human geimline may also be addressed.

The following approach may be used to determine the closest matching human germline for a given camelid VH (or VL) domain:

Before analyzing the percentage sequence identity between Camelidae and human germline VH and VL, the canonical folds may first be determined, which allows the identification of the family of human germline segments with the identical combination of canonical folds for H1 and H2 or L1 and L2 (and L3). Subsequently the human germline family member that has the highest degree of sequence homology with the Camelidae variable region of interest may be chosen for scoring sequence homology. The determination of Chothia canonical classes of hypervariable loops L1, L2, L3, H1 and H2 can be performed with the bioinformatics tools publicly available on webpage www.bioinf.org.uk/abs/chothia.html.page. The output of the program shows the key residue requirements in a datafile. In these datafiles, the key residue positions are shown with the allowed amino acids at each position. The sequence of the variable region of the antibody is given as input and is first aligned with a consensus antibody sequence to assign the Kabat numbering scheme. The analysis of the canonical folds uses a set of key residue templates derived by an automated method developed by Martin and Thornton (Martin et al., J. Mol. Biol. 263:800-815 (1996)). The boundaries of the individual framework regions may be assigned using the IMGT numbering scheme, which is an adaptation of the numbering scheme of Chothia (Lefranc et al., NAR 27: 209-212 (1999); http://imgt.cines.fr).

With the particular human germline V segment known, which uses the same combination of canonical folds for H1 and H2 or L1 and L2 (and L3), the best matching family member in terms of sequence homology can be determined. The percentage sequence identity between Camelidae VH and VL domain framework amino acid sequences and corresponding sequences encoded by the human germline can be determined using bioinformatic tools, but manual alignment of the sequences could also be used. Human immunoglobulin sequences can be identified from several protein data bases, such as VBase (http://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database (http://www.bioc.unizh.ch/antibody/Sequences/Germlines. To compare the human sequences to the V regions of Camelidae VH or VL domains a sequence alignment algorithm such as available via websites like www.expasy.ch/tools/#align can be used, but also manual alignment can also be performed with a limited set of sequences. Human germline light and heavy chain sequences of the families with the same combinations of canonical folds and with the highest degree of homology with the framework regions 1, 2, and 3 of each chain may be selected and compared with the Camelidae variable region of interest; also the FR4 is checked against the human germline JH and JK or JL regions.

Note that in the calculation of overall percent sequence homology the residues of FR1, FR2 and FR3 are evaluated using the closest match sequence from the human germline family with the identical combination of canonical folds. Only residues different from the closest match or other members of the same family with the same combination of canonical folds are scored (NB—excluding any primer-encoded differences). However, for the purposes of humanization, residues in framework regions identical to members of other human germline families, which do not have the same combination of canonical folds, can be considered for humanization, despite the fact that these are scored “negative” according to the stringent conditions described above. This assumption is based on the “mix and match” approach for humanization, in which each of FR1, FR2, FR3 and FR4 is separately compared to its closest matching human germline sequence and the humanized molecule therefore contains a combination of different FRs as was done by Qu and colleagues (Qu et la., Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Ono et al., Mol. Immunol. 36:387-395 (1999)).

Cross-competing Antibodies

Monoclonal antibodies or antigen-binding fragments thereof that “cross-compete” with the molecules disclosed herein are those that bind human CD70 at site(s) that are identical to, or overlapping with, the site(s) at which the present CD70 antibodies bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human CD70 can be hound to a solid support. Then, an antibody compound or antigen binding fragment thereof of the present invention and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such invention antibody compound are added. One of the two molecules is labelled. If the labelled compound and the unlabeled compound bind to separate and discrete sites on CD70, the labelled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labelled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labelled compound will bind. For purposes of the present invention, competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to CD70 by about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 99%. Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pages 567-569, ISBN 0-87969-314-2. Such assays can be made quantitative by using purified antibodies. A standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor. The capacity of an unlabeled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labelled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.

Preferred embodiments are antibodies which cross-compete for binding to human CD70 with antibodies comprising the llama-derived Fab 27B3, and its germlined variants, including in particular 41D12 (ARGX-110) and which exhibit the same combination of binding characteristics, namely:

-   -   (a) binding within the amino acid sequence HIQVTLAICSS (SEQ ID         NO:342) in human CD70;     -   (b) cross-reactivity with CD70 homologs of rhesus macaque         (Macaca mulatta) and cynomolgus monkey (Macaca cynomolgus);     -   (c) binding to native and heat denatured recombinant human CD70.         Polynucleotides Encoding CD70 Antibodies

The invention also provides polynucleotide molecules encoding the CD70 antibodies of the invention, also expression vectors containing a nucleotide sequences which encode the CD70 antibodies of the invention operably linked to regulatory sequences which permit expression of the antigen binding polypeptide in a host cell or cell-free expression system, and a host cell or cell-free expression system containing this expression vector.

Polynucleotide molecules encoding the CD70 antibodies of the invention include, for example, recombinant DNA molecules. The terms “nucleic acid”, “polynucleotide” or a “polynucleotide molecule” as used herein interchangeably and refer to any DNA or RNA molecule, either single- or double-stranded and, if single-stranded, the molecule of its complementary sequence. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5′ to 3′ direction. In some embodiments of the invention, nucleic acids or polynucleotides are “isolated.” This term, when applied to a nucleic acid molecule, refers to a nucleic acid molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or non-human host organism. When applied to RNA, the term “isolated polynucleotide” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been purified/separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated polynucleotide (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

In one embodiment, the invention provides nucleotide sequences which encode the VH domain and VL domain of the germlined llama-derived Fab denoted herein 41D12, wherein the VH domain has the amino acid sequence shown as SEQ ID NO:223 and the VH domain has the amino acid sequence shown as SEQ ID NO:241. In a preferred embodiment the nucleotide sequence encoding the VH domain having the amino acid sequence shown as SEQ ID NO:223 comprises the nucleotide sequence shown as SEQ ID NO:344, and the nucleotide sequence encoding the VL domain having the amino acid sequence shown as SEQ ID NO:241 comprises the nucleotide sequence shown as SEQ ID NO:345.

In one embodiment, the invention provides nucleotide sequences which encode the VH domain and VL domain of the germlined llama-derived Fab denoted herein 57B6, wherein the VH domain has the amino acid sequence shown as SEQ ID NO:274 and the VH domain has the amino acid sequence shown as SEQ ID NO:276. In a preferred embodiment the nucleotide sequence encoding the VH domain having the amino acid sequence shown as SEQ ID NO:274 comprises the nucleotide sequence shown as SEQ ID NO:346, and the nucleotide sequence encoding the VL domain having the amino acid sequence shown as SEQ ID NO:276 comprises the nucleotide sequence shown as SEQ ID NO:347.

In one embodiment, the invention provides nucleotide sequences which encode the VH domain and VL domain of the germlined llama-derived Fab denoted herein 59D10, wherein the VH domain has the amino acid sequence shown as SEQ ID NO:275 and the VH domain has the amino acid sequence shown as SEQ ID NO:277. In a preferred embodiment the nucleotide sequence encoding the VH domain having the amino acid sequence shown as SEQ ID NO:275 comprises the nucleotide sequence shown as SEQ ID NO:348, and the nucleotide sequence encoding the VL domain having the amino acid sequence shown as SEQ ID NO:277 comprises the nucleotide sequence shown as SEQ ID NO:349.

For recombinant production of a CD70 antibody according to the invention, a recombinant polynucleotide encoding it may be prepared (using standard molecular biology techniques) and inserted into a replicable vector for expression in a chosen host cell, or a cell-free expression system. Suitable host cells may be prokaryote, yeast, or higher eukaryote cells, specifically mammalian cells. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL 1581; ATCC CRL 8287) or NS0 (HPA culture collections no. 85110503); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), as well as DSM's PERC-6 cell line. Expression vectors suitable for use in each of these host cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to a cultured cell line. Whole human beings into which an expression vector encoding an antigen binding polypeptide according to the invention has been introduced are explicitly excluded from the definition of a “host cell”.

Antibody Production

In an important aspect, the invention also provides a method of producing a CD70 antibody of the invention which comprises culturing a host cell (or cell free expression system) containing polynucleotide (e.g. an expression vector) encoding the CD70 antibody under conditions which permit expression of the CD70 antibody, and recovering the expressed CD70 antibody. This recombinant expression process can be used for large scale production of CD70 antibodies according to the invention, including monoclonal antibodies intended for human therapeutic use. Suitable vectors, cell lines and production processes for large scale manufacture of recombinant antibodies suitable for in vivo therapeutic use are generally available in the art and will be well known to the skilled person.

As noted elsewhere, the CD70 antibodies provided herein, including in particular antibodies based on the Fab 27B3 and its germlined variants such as 41D12 (ARGX-110) display characteristics which are particularly beneficial for large-scale commercial manufacture. Specifically, the extremely high production yield (>4 g/L) achievable in a commercial scale recombinant expression system will dramatically reduce production costs.

Preferred embodiments also exhibit thermal stability when tested at 37° C. and also over several freeze-thaw cycles, which is again extremely beneficial for commercial manufacture and storage of a clinical product. Surprisingly, several of the human germlined variants of Fab 27B3, including 41 D12 and 40F1, exhibit improved thermal stability compared to 27B3 itself.

Accordingly, in a further aspect the invention provides an isolated antibody or antigen binding fragment thereof which binds to human CD70, said antibody or fragment comprising a heavy chain variable domain corresponding to the amino acid sequence set forth in SEQ ID NO:178, provided that at least one amino acid at a Kabat position selected from the group consisting of H6, H18, H24, H31, H56, H74, H74, H77, H79, H83, H84, H89, H93, H94, H108, H110, and H112 is substituted with another amino acid.

This antibody, or antigen binding fragment thereof, may exhibit greater thermal stability than an antibody, or antigen binding fragment thereof, comprising a heavy chain variable domain with the amino acid sequence set forth in SEQ ID NO:178.

In a further embodiment there is provided a CD70 antibody or antigen binding fragment which comprises a light chain variable domain corresponding to the amino acid sequence set forth in SEQ ID NO:201, provided that at least one amino acid at a Kabat position selected from the group consisting of L2, L11, L12, L25, L26, L30, L46, L53, L60, L61, L67, L68, L76, L80, L81, L85, and L87 is substituted with another amino acid.

This antibody, or antigen binding fragment thereof, may exhibit greater thermal stability than an antibody, or antigen binding fragment thereof, comprising a heavy chain variable domain with the amino acid sequence set forth in SEQ ID NO:201.

In a particular embodiment, the CD70 antibody, or antigen binding fragment thereof may comprise:

-   a) a heavy chain variable domain with the amino acid sequence set     forth in SEQ ID NO:178 comprising one or more amino acid     substitutions at Kabat positions selected from the group consisting     of H6, H18, H24, H31, H56, H74, H74, H77, H79, H83, H84, H89, H93,     H94, H108, H110, and H112; and -   b) a light chain variable domain with the amino acid sequence set     forth in SEQ ID NO:201 comprising one or more amino acid     substitutions at Kabat positions selected from the group consisting     of L2, L11, L12, L25, L26, L30, L46, L53, L60, L61, L67, L68, L76,     L80, L81, L85, and L87.

This antibody, or antigen binding fragment thereof, of claim 24, may exhibit greater thermal stability than an antibody, or antigen binding fragment thereof, comprising a heavy chain variable domain with the amino acid sequence set forth in SEQ ID NO:178 and a light chain variable domain with the amino acid sequence set forth in SEQ ID NO:201.

Therapeutic Utility of CD70 Antibodies

The CD70 antibodies, or antigen binding fragments thereof, provided herein can be used to inhibit the growth of cancerous tumour cells in vivo and are therefore useful in the treatment of CD70-expressing cancers.

Accordingly, further aspects of the invention relate to methods of inhibiting tumour cell growth in a human patient, and also methods of treating or preventing cancer, which comprise administering to a patient in need thereof a therapeutically effective amount of a CD70 antibody or antigen binding fragment as described herein.

Another aspect of the invention provides a CD70 antibody or antigen binding fragment as described herein for use inhibiting the growth of CD70-expressing tumour cells in a human patient.

A still further aspect of the invention provides a CD70 antibody or antigen binding fragment as described herein for use treating or preventing cancer in a human patient.

Preferred cancers whose growth may be inhibited using the CD70 antibodies described herein include renal cancer (e.g., renal cell carcinoma), breast cancer, brain tumors, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphomas (e.g., Hodgkin's and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), nasopharyngeal carcinomas, melanoma {e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, mesothelioma.

The CD70 antibodies described herein can also be used to treat a subject with a tumorigenic disorder characterized by the presence of tumor cells expressing CD70 including, for example, renal cell carcinomas (RCC), such as clear cell RCC, glioblastoma, breast cancer, brain tumors, nasopharangeal carcinomas, non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), Burkitt's lymphoma, anaplastic large-cell lymphomas (ALCL), multiple myeloma, cutaneous T-cell lymphomas, nodular small cleaved-cell lymphomas, lymphocytic lymphomas, peripheral T-cell lymphomas, Lennert's lymphomas, immunoblastic lymphomas, T-cell leukemia/lymphomas (ATLL), adult T-cell leukemia (T-ALL), entroblastic/centrocytic (cb/cc) follicular lymphomas cancers, diffuse large cell lymphomas of B lineage, angioimmunoblastic lymphadenopathy (AILD)-like T cell lymphoma, HIV associated body cavity based lymphomas, embryonal carcinomas, undifferentiated carcinomas of the rhino-pharynx (e.g., Schmincke's tumor), Castleman's disease, Kaposi's Sarcoma, multiple myeloma, Waldenstrom's macroglobulinemia, Mantle cell lymphoma and other B-cell lymphomas.

Specific embodiments relate to treatment of any one of the above-listed cancers with an antibody comprising the llama-derived Fab 27B3, or one of its germlined variants, including in particular 41D12 (ARGX-110). In particular, any one of the above-listed cancers may be treated using an antibody comprising the Fab regions of 41D12 (germlined variant of 27B3) fused to the constant regions of human IgG1. In the latter embodiment, the human IgG1 constant region may be further engineered in order to maximise antibody effector functions (e.g. by addition of point mutations), or the 41D12-IgG1 antibody may be non-fucosylated, again to enhance antibody effector function.

As noted elsewhere, the CD70 antibodies described herein, particularly 27B3 and its germlined variants, exhibit particularly strong binding to human cancer cell lines which express cell-surface CD70 at low copy number, such as for example the large B cell lymphoma cell line SU-DHL-6, the chronic lymphocytic leukemia cell line JVM-2, the cutaneous T cell lymphoma cell line HH, the gastric carcinoma cell line MKN-45, the lung carcinoma cell lines A549 and EBC-1, the melanoma cell lines WM852 and WM793, the glioblastoma cell line GaMG and the ovarian carcinoma cell lines OAW-42, SKOv3 and OVCAR3. Indeed, the affinity of 41D12 (ARGX-110) for these low copy-number cancer cell lines is significantly higher than comparator prior art antibodies, such as SGN70 and MDX1411.

The “improved” affinity for low copy-number cancer cell lines is of direct relevance for clinical treatment of the corresponding cancers, as demonstrated by the cell spiking experiments in the accompanying examples. When cells of a low copy-number cell line (e.g. the diffuse large B cell lymphoma cell line SU-DHL-6) are spiked into freshly isolated PBMCs from healthy donors, cells of the cancer cell line are preferentially depleted by the example CD70 antibody ARGX-110.

Accordingly, a further aspect of the invention relates to a method of treating or preventing cancer, which comprises administering to a patient in need thereof a therapeutically effective amount of a CD70 antibody or antigen binding fragment as described herein, wherein the cancer is a cancer which exhibits low copy-number expression of CD70.

The invention also provides a CD70 antibody or antigen binding fragment as described herein for use treating or preventing cancer in a human patient, wherein the cancer is a cancer which exhibits low copy-number expression of CD70.

In each of these aspects the low copy-number cancer is preferably selected from the group consisting of: large B cell lymphoma, chronic lymphocytic leukaemia, cutaneous T cell lymphoma, gastric cancer, lung cancer, melanoma, glioblastoma and ovarian cancer.

Specific embodiments relate to treatment of low copy-number cancers with an antibody comprising the llama-derived Fab 27B3, or one of its germlined variants, including in particular 41D12 (ARGX-110). In particular, any one of the above-listed low copy-number cancers may be treated using an antibody comprising the Fab regions of 41D12 (germlined variant of 27B3) fused to the constant regions of human IgG1.

The CD70 antibodies described herein are also useful for the treatment of immunological disorders characterised by expression of CD70. Specific examples of such immunological disorders include the following: rheumatoid arthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis, allergic encephalomyelitis), endocrine ophthalmopathy, uveoretinitis, systemic lupus erythematosus, myasthenia gravis, Grave's disease, glomerulonephritis, autoimmune hepatological disorder, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, Celiac disease), anaphylaxis, allergic reaction, Sjogren's syndrome, type I diabetes mellitus, primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis, subacute cutaneous lupus erythematosus, hypoparathyroidism, Dressier's syndrome, autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis herpetiformis, alopecia greata, pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyl), and telangiectasia), male and female autoimmune infertility, ankylosing spondolytis, ulcerative colitis, mixed connective tissue disease, polyarteritis ncdosa, systemic necrotizing vasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrent abortion, anti-phospholipid syndrome, farmer's lung, erythema multiforme, post cardiotomy syndrome, Gushing's syndrome, autoimmune chronic active hepatitis, bird-fancier's lung, toxic epidermal necrolysis, Alport's syndrome, alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lung disease, erythema nodosum, pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis, polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cell arteritis, ascariasis, aspergillosis, Sampler's syndrome, eczema, lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome, Kawasaki's disease, dengue, encephalomyelitis, endocarditis, endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum, psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus host disease, transplantation rejection, cardiomyopathy, Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia, Evan's syndrome, and autoimmune gonadal failure, disorders of B lymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, or graft versus host disease), or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupus erythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, or chronic graft versus host disease), Churg Strauss syndrome, microscopic polyangiitis and Takayasu's arteritis.

Specific embodiments relate to treatment of any one of the above-listed immunological disorders with an antibody comprising the llama-derived Fab 27B3, or one of its germlined variants, including in particular 41D12 (ARGX-110). In particular, any one of the above-listed cancers may be treated using an antibody comprising the Fab regions of 41D12 (germlined variant of 27B3) fused to the constant regions of human IgG1. In the latter embodiment, the human IgG1 constant region may be further engineered in order to maximise antibody effector functions (e.g. by addition of point mutations), or the 41D12-IgG1 antibody may be non-fucosylated, again to enhance antibody effector function.

As used herein, the term “treating” or “treatment” means slowing, interrupting, arresting, controlling, ameliorating, stopping, reducing severity of a symptom, disorder, condition or disease, but does not necessarily involve a total elimination of all disease-related symptoms, conditions or disorders.

For human therapeutic use the CD70 antibodies described herein may be administered to a human subject in need of treatment in an “effective amount”. The term “effective amount” refers to the amount or dose of a CD70 antibody which, upon single or multiple dose administration to a human patient, provides therapeutic efficacy in the treatment of disease. Therapeutically effective amounts of the CD70 antibody can comprise an amount in the range of from about 0.1 mg/kg to about 20 mg/kg per single dose. A therapeutic effective amount for any individual patient can be determined by the healthcare professional by monitoring the effect of the CD70 antibody on a biomarker, such as cell surface CD70 in tumour tissues, or a symptom such as tumour regression, etc. The amount of antibody administered at any given time point may be varied so that optimal amounts of CD70 antibody, whether employed alone or in combination with any other therapeutic agent, are administered during the course of treatment.

It is also contemplated to administer the CD70 antibodies described herein, or pharmaceutical compositions comprising such antibodies, in combination with any other cancer treatment, as a combination therapy.

Use of Antibodies which are Poorly Internalised to Deplete CD70-Expressing Cells

A surprising finding described herein is that CD70 antibodies bound to CD70 expressed on the surface of cancer cell lines are poorly internalised. This is a surprising result, since the prior art has previously described CD70 as an “internalising target”. Although there is variation between different cancer cell types in the precise degree of internalisation of bound CD70 antibodies, no cancer cell type is observed to “fully internalise” the bound CD70 antibody. For example, renal cancer cell lines previously described as rapidly internalising in fact are shown to internalise a maximum of about 70% of bound antibody ARGX-110. Many other cancer cell lines internalise less than 30%, less than 20% or even less than 10% of bound ARGX-110.

The variation in degree of internalisation between different cancer cell lines is largely disease-indication dependent, with similar results being observed using different CD70 antibodies. However, the poor internalisation in certain cell lines is particularly apparent when using the antibody ARGX-110. The observed low degree of internalisation on many cancer cells provides a rationale for the use of CD70 antibodies with strong effector function in order to deplete (e.g. kill or inhibit the growth of) cancer cells in vivo. Since bound CD70 antibodies are relatively poorly internalised on many cancer cells, the antibody will remain hound to the cell surface and can thereby trigger effector functions (ADCC, CDC, ADCP) by interactions with the immune system of a human patient.

Accordingly, a further important aspect of the invention relates to a method of depleting CD70-expressing cells in a human patient, comprising administering to said patient an effective amount of an antibody which binds to human CD70, wherein said CD70-expressing cells exhibit 30% or less, 25% or less, 20% or less, 15% or less, 10% or less or 5% or less internalisation of said antibody after a period of 6 hours, and wherein said antibody exhibits at least one effector function selected from the group consisting of ADCC, CDC and ADCP.

In this method, the CD70 antibody (with active effector functions) is used to deplete CD70 expressing cells. This depletion of CD70-expressing cells may form the basis of therapeutic or prophylactic treatment, e.g. for treatment of prevention of CD70-expressing cancers, or for treatment of prevention of CD70-associated immunological disorders. The precise mechanism by which CD70-expressing antibodies are depleted may depend on the type of effector functions exhibited by the CD70 antibody, but will be dependent on strong (high affinity) binding of the CD70 antibody to cell-surface CD70, coupled with poor internalisation of the bound antibody.

In certain embodiments the CD70-expressing cells are CD70-expressing cancer cells. In particular, the CD70-expressing cancer cells may be of a cancer type selected from the group consisting of: Burkitt lymphoma, large B cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, pancreatic carcinoma, gastric carcinoma, glioblastoma and lung carcinoma. Cell lines derived from each of these cancers have been demonstrated to internalise 30% or less of bound CD70 antibody over a period of 6 hours. In a further embodiment CD70-expressing cancer cells may be of a cancer type selected from the group consisting of: Burkitt lymphoma, large B cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, gastric carcinoma, glioblastoma and lung carcinoma. Cell lines derived from each of these cancers have been demonstrated to internalise 20% or less of bound CD70 antibody over a period of 6 hours. In a further embodiment CD70-expressing cancer cells may be of a cancer type selected from the group consisting of: Burkitt lymphoma, large B cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, gastric carcinoma and lung carcinoma. Cell lines derived from each of these cancers have been demonstrated to internalise 10% or less of bound CD70 antibody over a period of 6 hours.

In other embodiments the CD70-expressing cells may be CD70-expressing activated T cells, which do not exhibit any detectable internalisation of bound CD70 antibodies (specifically no internalisation of ARGX-110 is observed over a period of 6 hours. This observation that bound CD70 antibodies are not internalised by CD70+ activated T cells is strongly supportive of the use of CD70 antibodies with strong effector function (ADCC, CDC or ADCP), including but not limited to ARGX-110, in the treatment of immunological diseases mediated by CD70+ activated T cells, including autoimmune disorders such as for example rheumatoid arthritis, psoriasis, SLE, etc.

The above-listed embodiments can utilise essentially any CD70 antibody. In specific embodiments the antibody can be any CD70 antibody which has been shown experimentally to exhibit 30% or less, 25% or less, 20% or less, 15% or less, 10% or less or 5% or less internalisation after a period of 6 hours in a CD70-expressing cancer cell line selected from the group consisting of Raji, SU-DHL-6, MHHPREB1, Mino, Mec1, JVM-2, MKN-45, A549, U87MG, PANC-1, PANC-89, L428, Granta 519, Rec-1 and EBC-1. In effect, the cell line internalisation assay described in the accompanying examples can be used as a screen both to determine whether particular disease indications should be targeted with CD70 antibodies having strong effector function, or with CD antibody-drug conjugates (discussed below), and also to identify suitable CD70 antibodies for use in treating particular disease indications.

Accordingly, the invention also provides a method of depleting CD70-expressing cancer cells in a human patient, comprising administering to said patient an effective amount of an antibody which binds to human CD70, wherein said antibody exhibits 30% or less internalisation after a period of 6 hours in a CD70-expressing cancer cell line selected from the group consisting of Raji, SU-DHL-6, MHHPREB1, Mino, Mec1, JVM-2, MKN-45, A549, U87MG, PANC-1, PANC-89, L428, Granta 519, Rec-1 and EBC-1, and wherein said antibody exhibits at least one effector function selected from the group consisting of ADCC, CDC and ADCP. In one embodiment the antibody to be used to deplete CD70-expressing cancer cells in a human patient may be an antibody which exhibits 10% or less internalisation over a period of 6 hours in a CD70-expressing cancer cell line selected from the group consisting of Raji, SU-DHL-6, Granta 519, Rec-1, Mec1, JVM-2, MKN-45, A549 and EBC-1, and also exhibits at least one effector function selected from the group consisting of ADCC, CDC and ADCP.

Specific embodiments of this aspect of the invention may utilise any of the CD70 antibodies described herein, which bind human CD70 with extremely high affinity. Preferred embodiments may utilise an antibody comprising the llama-derived Fab 27B3, or one of its germlined variants, including in particular 41D12 (ARGX-110). Particularly preferred embodiments utilise an antibody comprising the Fab regions of 41D12 (germlined variant of 27B3) fused to the constant regions of human IgG1. In the latter embodiment, the human IgG1 constant region may be further engineered in order to maximise antibody effector functions (e.g. by addition of point mutations), or the 41D12-IgG1 antibody may be non-fucosylated, again to enhance antibody effector function.

Since the rationale behind the use of poorly internalising CD70 antibodies to deplete CD70-expressing cells relies on antibody effector functions, it is preferred that the antibodies used in this aspect of the invention are not immunoconjugates in which the CD70 antibody is linked to a pharmaceutical agent, cytotoxic agent, cytostatic agent, drug moiety, etc. Indeed, a particular advantage of this aspect of the invention is avoidance of the need to use antibody-drug conjugates, which is desirable both from a patient-safety perspective (avoids administration of potentially toxic agents) and an economic perspective (avoids costly synthesis of antibody-drug conjugates).

Use of Antibody-drug Conjugates to Kill Cells with Significant Internalisation CD70 Antibodies

It is demonstrated in the present examples that there are significant cell-to-cell differences in the degree of internalisation of CD70 antibodies, and that these differences are largely dependent on cell type, rather than the nature of the CD70 antibody (although differences are particularly marked when using the antibody ARGX-110). For those cell types which exhibit a significant degree of internalisation of bound CD70 antibody, i.e. 30% or more, preferably 40% or more, more preferably 50% or more, or even 60% or more internalisation after 6 hours, an altenative therapeutic strategy may be based on the use of CD70 antibody-drug conjugates.

Accordingly, a further important aspect of the invention relates to a method of depleting CD70-expressing cells in a human patient, comprising administering to said patient an effective amount of an antibody-drug conjugate comprising an antibody which binds to human CD70 and a cytotoxic or cytostatic drug moiety, wherein said CD70-expressing cells exhibit 30% or more, 40% or more, 50% or more, or 60% or more internalisation of said antibody which binds to human CD70 after a period of 6 hours.

In specific embodiments the CD70-expressing cells may internalise between 40% and 70%, preferably between 50% and 70% of the CD70 antibody which forms the basis of the antibody-drug conjugate within a period of 6 hours.

In this method, the CD70 antibody-drug conjugate is used to deplete CD70 expressing cells. This depletion of CD70-expressing cells may form the basis of therapeutic or prophylactic treatment, e.g. for treatment of prevention of CD70-expressing cancers, or for treatment of prevention of CD70-associated immunological disorders. The precise mechanism by which CD70-expressing antibodies are depleted may depend on the nature of the antibody-drug conjugate, i.e. whether the “drug” moiety is cytotoxic or cytostatic, but will be dependent on strong (high affinity) binding of the CD70 antibody to cell-surface CD70, coupled with significant internalisation of the bound antibody.

In certain embodiments the CD70-expressing cells are CD70-expressing cancer cells. In particular, the CD70-expressing cancer cells may be of a cancer type selected from the group consisting of: cutaneous T cell lymphoma, multiple myeloma, renal cell carcinoma, astrocytoma, melanoma and ovarian carcinoma. Cell lines derived from each of these cancers have been demonstrated to internalise more than 30% of bound CD70 antibody over a period of 6 hours. In a further embodiment, the CD70-expressing cancer cells may be of a cancer type selected from the group consisting of: renal cell carcinoma, astrocytoma, melanoma and ovarian carcinoma. Cell lines derived from each of these cancers have been demonstrated to internalise more than 50% of bound CD70 antibody over a period of 6 hours.

In specific embodiments of this aspect of the invention, the antibody-drug conjugate may be based on any of the CD70 antibodies described herein, which bind human CD70 with extremely high affinity. In preferred embodiments the antibody-drug conjugate may be based on an antibody comprising the llama-derived Fab 27B3, or one of its germlined variants, including in particular 41D12. The features of antibody-drug conjugates intended for human therapeutic use are generally known in the art, particularly with regard to the nature of the “drug” moiety, e.g. a cytotoxic or cytostatic agent, and means of conjugation of the drug moiety to the CD70 antibody moiety. Examples of CD70 antibody-drug conjugates are given, for example, in WO 2004/073656.

Pharmaceutical Compositions

The scope of the invention includes pharmaceutical compositions, containing one or a combination of CD70 antibodies of the invention, or antigen-binding fragments thereof, formulated with one or more a pharmaceutically acceptable carriers or excipients. Such compositions may include one or a combination of (e.g., two or more different) CD70 antibodies.

Techniques for formulating antibodies for human therapeutic use are well known in the art and are reviewed, for example, in Wang et al., Journal of Pharmaceutical Sciences, Vol. 96, pp 1-26, 2007.

Incorporation by Reference

Various publications are cited in the foregoing description and throughout the following examples, each of which is incorporated by reference herein in its entirety.

EXAMPLES

The invention will be further understood with reference to the following non-limiting experimental examples.

Example 1 Immunization of Llamas

Immunizations of llamas and harvesting of peripheral blood lymphocytes (PBLs) as well as the subsequent extraction of RNA and amplification of antibody fragments were performed as described by De Haard and colleagues (De Haard H, et al., J. Bact. 187:4531-4541, 2005). Two llamas were immunized with CD70-expressing 786-O cells (ATCC-CRL-1932) and two llamas with CD70-expressing Raji cells (ATCC-CCL-86). Cells were prepared freshly for each immunization and were verified for CD70 expression by FACS analysis. The llamas were immunized with approximately 10⁷ live cells injected intramuscularly in the neck, once per week for six weeks. Freund's Incomplete Adjuvant was injected in the neck muscles a few centimetres away from the site of cellular immunization.

Blood samples of 10 ml were collected pre- and post immunization to investigate the immune response. The sera from the llamas were tested for the presence of antibodies against recombinant CD70 (Flag-TNC-CD70) by ELISA prior to (day 0) and after (day 55 or 69) immunization. It should be noted that as detection antibody the goat anti-llama IgG1/2 (Bethyl, A160-100P) was used that does not discriminate between conventional and heavy chain antibodies, meaning that the measured CD70 response is from the total IgG. The results are shown in FIG. 1.

Three-to-four days after the last immunization, 400 ml blood was collected for extraction of total RNA from the PBLs using a Ficoll-Paque gradient to isolate PBLs and the method described by Chomczynski P, et al., Anal. Biochem. 162: 156-159, 1987 to prepare the RNA. In average, RNA yields of 450 μg were achieved, of which an 80 μg aliquot was used for random cDNA synthesis and subsequent amplification of VHCH1, VλCλ and VκCκ gene segments.

Example 2 Library Construction

Independent VλCλ, and VκCκ libraries were constructed using a two step PCR, in which 25 cycles with non tagged primers was done followed by 10 cycles using tagged version of these primers (De Haard H, et al., Biol. Chem. 274, 1999). The VHCH1 libraries were built in parallel using the same approach. The sizes of the individual libraries were between 10⁷ and 10¹⁰ cfu. Next, the light chain from the VλCλ and VκCκ libraries were re-cloned separately in the VHCH1-expressing vector to create the “Lambda” and “Kappa” llama Fab-library respectively. Alternatively the digested VHCH1 amplicons were directly cloned into the VκCκ and VλCλ libraries avoiding the construction of a separate VHCH1 library. As light chain shuffling generally delivers better affinity variants than heavy chain shuffling does, we chose to generate precloned light chain libraries and directly clone the heavy chain amplicons in these primary repertoires.

The final phage display libraries were between 5×10⁸ and 2×10⁹ cfu. Quality control of the libraries by analysis of percentage of full length Fab containing clones was routinely performed using PCR.

Example 3 Selections and Screening

Two-to-three rounds of phage selections were done on either captured recombinant CD70 (Flag-TNC-CD70) or on directly coated CD70 using standard protocols. Elution of bound phage was done with trypsin.

From selected phage transfected into TG1, individual colonies were picked randomly for growth in 96 well plates. After IPTG induction according to standard protocols periplasmic fractions (peris) containing Fabs were prepared. In all selections and for all four llamas, several clones were found that were able to express Fabs capable in inhibiting the interaction between CD27 and CD70 as determined by the inhibition ELISA (FIGS. 2 and 3). In this assay, Flag-TNC-CD70 is captured by an anti-Flag mAb and binding of CD27-Fc is detected via a biotinylated anti-CD27 mAb and strep-HRP in the absence or presence of CD70 Fabs (FIGS. 2 and 3) or CD70 mAbs (FIG. 4). In FIG. 2B it can be seen that 27B3, which has an identical VH as 9D1, but contains a somewhat different light chain, seems to block binding of CD27 to CD70 more efficiently, which was in line with the better off rate of this Fab as measured on Biacore (data not shown). Table 3 indicates the blocking potency (expressed as IC50) for the various llama-human chimeric mAbs tested in the CD70 inhibition ELISA. Fab 27B3 has an IC50 of around 0.4 nM, which even seems to improve upon formatting into a chimeric llama—human IgG1 yielding an IC 50 of 0.25 nM, whereas mAb 9D1 with the other light chain is 3-fold less potent (0.73 nM). FIG. 3 is a composite of an experiment testing llama CD70 Fabs for blocking activity (in the inhibition ELISA, see white bars) and for CD70 binding (binding ELISA, black bars). For the binding ELISA, Flag-TNC-CD70 is captured by an anti-Flag mAb and binding of the Fabs is detected using an anti-myc-HRP mAb, which recognizes the MYC tag fused to the C-terminus of the Fd fragment. For comparison purposes, known monoclonal antibodies to CD70 (SGN70 described in US 2010/0129362 A1, clone 1F6; and MDX69A7 described in WO2008/074004 FIGS. 5A and 5B) were tested in parallel. Many llama-derived Fab clones were identified that demonstrate pure binding in ELISA and are non-blocking (see FIG. 3—for example Fab clones 45H8, 45C4, 46A2, 46G7, 46E4, 46F12 and 46G12). Of interest is clone 59D10, which as Fab has no blocking activity, but when converted into a chimeric IgG was antagonistic (see Table 3). Amino acid sequences of the VH and VL domains of binding and blocking llama-derived Fab clones are shown elsewhere herein (Tables 6-9).

TABLE 3 Blocking potency expressed as IC50 (ng/ml) for individual SIMPLE antibodies (mAbs) and benchmarks SGN70 and MDX69A7 as measured in the CD70 inhibition ELISA using recombinant human CD70. mAb IC50 (ng/ml) SGN70 330 MDX69A7 4038 9D1 109 5F4 296 9E1 1400 9G2 40 5B2 92 9B2 126 4D2 254 24D4 84 24B6 420 27B3 38 19G10 75 57B6 154 59D10 107 mAbs 27B3 and 57B6 was also tested for their ability to inhibit the interaction between rhesus recombinant CD70 (FLAG-TNC-rhesus CD70) and CD27. The IC50 value determined by the inhibition ELISA described above was 150 and 135 ng/ml, respectively.

Example 4 Affinity for Human and Rhesus CD70

Off-rates of the purified llama-derived anti-CD70 blocking Fabs were determined using the Biacore. Recombinant human CD70 was immobilized on a CM5 Biacore chip. The immobilization was performed in accordance with a method provided by Biacore and by using the NHS/EDC kit (Biacore AB): after activation of the chip, a solution of 50 μg/ml of recombinant CD70 in 10 mM acetate buffer with pH of 5 was prepared and 1 μl of this solution (50 ng) was injected resulting in a surface density of approximately 1000 RU.

Fabs for the CD70 mAbs ARGX-110, MDX2H5 and SGN70 were prepared by trypsin digestion (SGN70 and MDX69A7 are described elsewhere herein, MDX2H5, also referred to as MDX1411, is described in WO2008/074004 FIGS. 1A and 1B). Fabs, at a concentration of approximately 100-400 μg/ml, were diluted 6-fold in hepes-buffered saline (0.1M Hepes, 1.5M NaCl, 30 mM EDTA, 0.5% v/v surfactant P20). They were injected (30 μl) and passed through the flow cells at a flow rate of 30 μl/min. After binding of the Fab to CD70, the off-rate was monitored for a period of 10 minutes. After the dissociation, the flow cell surfaces were regenerated by injecting 5 μl of 10 mM NaOH. Sometimes multiple injections of NaOH were needed to regenerate the surfaces depending on the affinity of the Fabs. Off-rate analysis was done by applying the BIAevaluation software. First, the sensogram of the blank runs were subtracted from those obtained with the coated flow cell. Then the off-rate was determined for a time range of 10 minutes using the Fit kinetics application and the K_(d) value was calculated. Moreover, the off-rate of CD27 for CD70 was determined as well. The off rates are summarized in Table 4.

All llama-derived Fabs tested bind with apparent very low off-rates (i.e. high affinity) to human recombinant CD70 and to rhesus recombinant CD70 based on Biacore off rates in the range of 0.4−4.8×10⁻⁴ s⁻¹. The off-rates of the llama-derived Fabs for human CD70 are much better than for the reference mAb derived Fabs (7-30×10⁻⁴ s⁻¹). The llama-derived Fabs with the best off-rates were shown to have comparable off-rates to that of CD27 for its interaction with CD70 (0.8×10⁻⁴ s⁻¹). It should be noted that the lower limit for measurement of off-rates using Biacore is around 0.4×10⁻⁴ s⁻¹. Hence the llama-derived Fabs with off-rates falling close to this limit as assessed by Biacore may in fact exhibit even a higher affinity for CD70.

TABLE 4 Off rates of llama-derived Fabs and reference Fabs for human or rhesus CD70 k_(off) (s⁻¹) × E⁻⁴ k_(off) (s⁻¹) × E⁻⁴ Fab tested human CD70 rhesus CD70 SGN70-Fab 7.0 MDX2H5-Fab 17 No binding MDX69A7-Fab 30 CD27 0.8 1C2 0.4 3.2 9D1 4.8 2.0 9G2 1.8 1.8 5F4 0.8 5.0 9E1 1.9 2.0 9B2 2.8 6.1 4D2 3.3 2.9 27B3 0.8 24E3 0.8 33D8 4.5 24F2 1.6 24B6 1.0 19G10 0.8 8B12 3.6 45B12 2.1 45D9 2.1 45F8 2.0 57B6 1.0 0.4 59D10 13.9 No binding

Example 5 Binding of Chimeric Llama-human CD70 mAbs to CD70 Positive Cells

The VH and VL of interesting llama-derived Fab clones were fused to constant regions of human IgG1 and to human Cλ (all variable light chain regions are of lambda origin) and produced as bivalent chimeric monoclonal antibodies in the system described in patent application WO 2009/145606. The chimeric llama-human mAbs were purified on Protein A followed by gel filtration.

786-O cells were incubated with a 1/5 dilution series of chimeric llama-human CD70 inhibiting mAbs (20 μg/ml-0.25 ng/ml) and binding of the mAbs to the cells was detected using an anti-human Fc-FITC antibody (1/500 diluted conjugate AF006 from supplier Binding Site). Fluorescence of 10,000 cells/condition was measured using a flow cytometer and the median fluorescence was plotted. The results are shown in FIG. 5. Most of the mAbs bind with high affinity to 786-O cells. SIMPLE antibody 1C2, which as Fab showed to have the best off rate and blocking potency, was not able to recognize cell surface expressed CD70 (data not shown) and therefore was not studied furthermore. SIMPLE antibody 27B3 has the best affinity for cell bound receptor with an EC50 of 0.24 nM.

In a further experiment, NHL-derived MHH-PREB-1 cells (1W cells) were incubated with a concentration gradient of chimeric llama-human CD70 inhibiting mAbs (20 μg/ml-0.25 ng/ml) and binding of the mAbs to the cells was detected using anti-human IgG-FITC antibody. The results are shown in FIG. 5B. The EC50 values for 57B6 and 59D10 were 83 and 74 ng/ml, respectively.

Example 6 Inhibition in Co-culture Experiments by Chimeric Llama-human CD70 mAbs

In order to determine the blocking potency in a cell based assay the system described by Wyzgol and colleagues was used (J Immunol (2009) 183: 1851-1861). Fibrosarcoma cell line HT1080 transfected with human CD27 secretes IL-8 upon ligation with trimeric recombinant CD70 and blocking of this interaction with a neutralizing mAb can be measured by reduced cytokine levels. A modified version of the assay was applied in which the B cell lymphoma Raji cell line served as source of CD70, although the molecule is expressed on the cell surface and therefore is more relevant than soluble CD70.

Raji cells were cultured and checked for CD70 expression by FACS analysis. Cells were then mixed in a 96-well tissue culture plate (50,000 cells/well) with the anti-CD70 mAbs and incubated at RT for 1 hour. HT1080-CD27 cells were added (10,000 cells/well) and thoroughly mixed. Co-cultures were transferred to the incubator and grown overnight at 37′C. Supernatants were collected and analyzed for their IL-8 content by ELISA. The results for the SIMPLE antibodies are shown in FIG. 6 and are compared to reference CD70 mAbs. IC50 values for the chimeric llama-human CD70 mAbs are between 3 and 517 ng/ml as compared to 42 ng/ml for SGN70 and 143 ng/ml for MDX69A7 (see table 5). SIMPLE antibodies 27B3 (IC50 of 33 pM) and 9G2 (IC50 of 20 pM) are 10 to 20 fold more potent than benchmark SGN70 (IC50 of 370 pM) that again is 3 fold more potent than benchmark MDX69A7 (1 nM). It can therefore be concluded that the chimeric llama-human mAbs are extremely potent in blocking the interaction between CD27 and CD70 and outperform the benchmark antibodies in this highly relevant bioassay.

TABLE 5 Neutralizing potency expressed as IC50 value (ng/ml) of chimeric llama-human CD70 mAbs and reference mAbs tested in Raji-based co-culture assay mAb IC50 (ng/ml) 9D1 29 5F4 32 9B2 213 4D2 54 9E1 517 5B2 19 SGN70 55 MDX69A7 143 27B3 5 19G10 16 9G2 3 57B6 5 59D10 10

Example 7 Antibody Dependent Cellular Cytoxicity Activity

Antibody Dependent Cellular Cytoxicity (ADCC) was measured using the standard Cr⁵¹-release assay, which was described by McEarchern et al. (Blood (2007) 109: 1185-1192). Human peripheral blood mononuclear cells (PBMC) were purified from heparinized whole blood by standard ficoll separation and were used as source of NK cells (i.e. effector cells). Blood from several independent donors was used. The cells were suspended at 2×10⁶/ml in media containing 200 U/ml of human IL-2 (for stimulation of NK cells) and incubated overnight at 37° C. The following day, adherent and non-adherent cells were collected and washed once in culture media. The target cells were 786-O cells (RCC) and were labeled with Cr⁵¹. Target to effector cell ratio was 1:20 and the incubation was performed for 2 hours with the antibody present at different concentrations in the culture medium. Chimeric llama-human mAbs as well as the benchmark antibodies were tested for ADCC activity on 786-O cells at different concentrations.

The percent lysis was determined by the equation: % Lysis=(sample CPM−spontaneous release CPM)/(maximum release CPM−spontaneous release CPM)×100.

The results demonstrate that all of the chimeric llama-human mAbs induce lysis of target cells via ADCC activity (FIG. 7). SIMPLE mAb 27B3 has a potency of around 1 μg/ml and therefore is 10 fold more potent than the affinity variant 9D1 that has a comparable IC50 as benchmark SGN70, which again is 2.5 fold more potent in ADCC than benchmark MDX69A7. SIMPLE antibody 27B3 combines best in class receptor—ligand blocking potency with superior ADCC potency and therefore has a favourable therapeutic profile.

Example 8 Complement Dependent Cytoxicity Potency of CD70 Specific mAbs

Complement Dependent Cytotoxicity (CDC) properties of the antibodies were determined in the standard assay described by McEarchern et al. (Blood (2007) 109: 1185-1192). In a first control experiment, U266 (MM) and MHH-PREB1 (NHL) cells were tested for the presence of CD70 antigen by FACS analysis. In a next experiment, a chimeric llama-human CD70 mAb was tested for CDC activity on both cell lines at 3 different concentrations and in the presence of 3, 6 or 9% human serum (as a source of human complement). It was concluded that the U266 cells give the best signal/noise ratio in the presence of 9% serum.

In a next experiment, U266 cells were mixed with chimeric llama-human mAbs (FIG. 8, upper panel) or the reference CD70 mAbs (FIG. 8, lower panel) at 0.001-20 μg/ml in the absence or presence of 9% human serum and incubated for 2 h at 37° C. Cells were spun down and Propidium Iodide was added to determine the number of dead cells. Cell lysis was measured in FACS. CDC activity was demonstrated for all chimeric llama-human mAbs on U266 cells in the presence of 9% human serum. SIMPLE antibody 27B3 has a similar IC50 as benchmark SGN70, while again MDX69A7 is 10 fold less potent.

Example 9 Antibody Dependent Cellular Phagocytosis Potency

To analyze Antibody Dependent Cellular Phagocytosis (ADCP) properties of the SIMPLE mAbs the assay described by McEarchern et al. (Blood (2007) 109: 1185-1192) was implemented. In this assay human peripheral blood mononuclear cells (PBMC) were purified from heparinized whole blood by standard ficoll separation. Blood from different donors were used. Cells were incubated in RPMI containing 10% FCS in tissue culture flask overnight at 37° C. Non-adherent cells were removed and adherent cells were cultured for 15 days in 75 ml X-VIVO 15 medium containing 500 U/ml rhGM-CSF (7.5 μl/75 ml). After 15 days, adherent cells were harvested (monocyte derived macrophages (MDM).

8.0×10⁴ target cells (786-O cells) loaded with the red dye PKH26 were incubated with mAb for 30 minutes on ice in a V-bottom 96 well plate. Next, 2.0×10⁴ MDM cells were added (target to effector (786-O to MDM) ratio of 4:1) and after 1 hour of incubation at 37° C., the cells were centrifuged, resuspended in 100 μl PBS 1% BSA and transferred to a V-bottom 96 well plate. Anti-CD11b monoclonal antibody (FITC conjugated) for staining of macrophages was added for 30 min while keeping the plates on ice, cells were then washed two times with PBS and fixed with 1% paraformaldehyde (in PBS). Samples were analysed by FACS, where phagocytosis was scored on double stained cells. The results are shown in FIG. 9 and these demonstrate that all chimeric llama-human mAbs tested are potent in inducing ADCP. SIMPLE antibody 27B3 induces phagocytosis of up to 50% of the labelled tumour cells by macrophages at concentrations of 1 μg/ml with an IC50 of around 100 ng/ml (0.4 nM).

Example 10 Internalization of Anti-CD70 mAbs on Tumor Cell Line 786-O

It has been demonstrated that binding of CD70 antibodies to the renal carcinoma derived cell line 786-O results in the rapid (within 1 hour) internalization of the antibody-receptor complex (Adam et al., Br J Cancer (2006) 95:298-306). In order to test for internalization of SIMPLE antibodies and benchmark mAbs directed against CD70, 786-O cells were cultured in a 96-well microtiter plate and incubated overnight at 37° C. 2.5 μg/ml mAb was added and incubated with the cells for 0-24 hours at 37° C. Plates were washed 3 times 5′ with stripping buffer (150 mM NaCl, 100 mM Glycine, pH=2.5; coded “IN” representing the amount of mAb internalized via CD70) or PBS (coded “OUT” and representing the amount of mAb bound to the receptor at the outside of the cell). Subsequently, cells were fixed with 4% paraformaldehyde for 30′ at RT, washed with PBS, and incubated 5′ with 0.2% Triton-X-100 (“IN”) or PBS (“OUT”). Next, cells were washed twice and incubated at RT for 10′ with 100 mM glycine followed by a 30′ incubation with PBS+1% BSA. Finally cells were stained with goat anti-human Fc (Jackson immunoresearch 109-005-098) and anti-goat IRDYE800 (Li-cor 926-32214) before analysis on the L1-Cor Odyssey infrared scanner. The antibody remaining at the outside of the cell, i.e. non-internalized, is scored as mean MFI OUT in FIG. 10. The results demonstrate that several mAbs remain longer at the surface of the cells than the other mAbs (higher signal). However, none of the mAbs internalizes completely: initially (between 0 and 2 hours), mAb internalization goes very fast, but then it seems that a steady state is reached where 30-40% of the mAb remains at the outside of the cell, even after 24 hours of incubation at 37° C. SIMPLE antibody 27B3 internalizes as rapidly as benchmark antibodies SGN70 and MDX69A7. It is interesting to observe that SIMPLE antibodies 9E1 and 19G10, but also other, remain at high levels at the outside of the cells, even after 24 hours of incubation (lower panel of FIG. 10). In order to effectively induce ADCC, CDC and ADCP effects it is important that high levels of CD70 specific antibodies remain on the outside of the targeted cancer cells in order to recruit the effector immune cells that are responsible for cell killing. On the other hand rapidly internalizing antibodies have potential as Antibody Drug Conjugates (ADC).

Example 11 In vivo Efficacy of Chimeric Llama-human mAbs in a Tumour Xenograft Model

To establish disseminated disease, 10⁶ Raji cells in 0.2 mL PBS were injected into the lateral tail vein of C.B.-17 severe combined immunodeficient (SCID) mice (Harlan, Indianapolis, Ind.). After injection, all of the mice were pooled and then placed randomly into the various treatment groups, with 9 mice per group. Antibodies were administered in 0.5 ml at day 1, 4, 8, 11, 15 after the cell implantation by intraperitoneal injection. Mice were monitored at least twice per week and were sacrificed when they exhibited signs of disease, including weight loss of 15% to 20%, hunched posture, lethargy, cranial swelling, or dehydration. Mice received treatment with 41D12 mAb in a dose response. Additionally, a Fc-dead version of 41D12 was tested at 10 mg/kg. This Fc-dead version has been described before in the literature and has no ADCC and CDC activity and much lower ADCP activity (McEarchern et al., Clin Cancer Res (2008) 14(23):7763-72). Results are shown as survival curves in FIG. 11. These data show that the median survival time for the control mice is 27 days. Median survival time was prolonged to at least 67 days when doses equal to or higher than 0.1 mg/kg of 41D12 were administered. The dose of 0.01 mg/kg is still efficacious. The Fc-dead version of 41D12 has no potency in vivo, illustrating the important of ADCC, CDC and ADCP in this model.

Example 12 Expression of Non-fucosylated mAbs

Antibodies with reduced amounts of fucosyl residues have been demonstrated to increase ADCC by 10-1000 fold (Iida et al., Clin Cancer Res. (2006) 12(9): 2879-2887). The CHO cell line Ms704-PF and MS705, which lack the fucosyltransferase gene (FUT8, BioWa, Inc.) was electroporated with a eukaryotic expression vector encoding the heavy and light chain of the chimeric llama-human CD70 mAbs. Drug resistant clones were selected by growth in Excell 325-PF CHO medium. Clones were screened for IgG production by a standard CD70 binding ELISA.

TABLE 6 sequence of CD70 specific llama-derived VH SEQ SEQ Fab Framework 1 ID CDR1 ID 1C2 ELQVVESGGGLVQPGGSLRLSCAASGFTLS   1 NYWMH  10 9D1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 8B12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 8C12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 9E1 EVQLQESGGGVVQPGGSLRLSCAASGFTFD   3 DYAMS  12 5F4 EVQVQESGGGLVHPGGSLKLSCAASGFTFD   4 TYAMS  13 5B2 QVQLVESGGDLVQPGGSLRLSCAASGFTVS   5 NPAMS  14 6D5 EVQLVQPGAELRKPGASVKVSCKASGYTFT   6 SYYID  15 4D2 QVQLQESGPGLVKPSQTLSLACTVSGGSIT   7 TSYYYWS  16 9A1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 SYAMS  17 9G2 QVQLVESGGGLMQPGGSLRLSCAASGFTFS   8 SSAMS  18 9B2 QLQVVESGGGLMQPGGSLRLSCAASGFTFS   9 GSAMS  19 27B3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 24E3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 33D8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 24F2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 VYYMN  11 24B6 EVQLQESGGGVVQPGGSLRLSCAASGFTFD   3 DYAMS  12 19G10 EVQLQESGGGVVQPGGSLRLSCAASGFTFD   3 DYAMS  12 45B12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 AYYMN  20 45D9 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 AYYMN  20 45F8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 AYYMN  20 45A12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 AYYMN  20 45B6 EVQLVESGGGLVQPGGSLRLSCAASGFTFS   2 AYYMN  20 57B6 QLQLVESGGGLVQPGGSLRLSCAASGFSFS 254 HYAMS 256 59D10 EVQLVESGGGLVQPGGSLRLSCAASELSFS 255 ISEMT 257 SEQ SEQ Fab Framework 2 ID  CDR2 ID 1C2 WVRQAPRKGLEWVS  21 TISTDNSRTYYADSVKG  26 9D1 WVRQAPGKGLEWVS  22 DINNEGGTTYYADSVKG  27 8B12 WVRQAPGKGLEWVS  22 DINNEGDTTYYADSVKG  28 8C12 WVRQAPGKGLEWVS  22 DINNEGDTTYYADSVKG  28 9E1 WVRQAPGKGLEWVS  22 SIYMYDSSTYYADSVKG  29 5F4 WVRQAPGKGLEWVS  22 AISWSGGETFYAESMKG  30 5B2 WVRQAPGKGLEWVS  22 EITNYGYNRYYADSVKG  31 6D5 WVRQAPGQGLEWMG  23 RIDPEDGGTKYAQKFQG  32 4D2 WIRQPPGKGLEWMG  24 AIGSRGSTYYSPSLKT  33 9A1 WVRQAPGKGLEWVS  22 DINSGGGSTKYNDSVKG  34 9G2 WVRQAPGKGLEWVS  22 SIYSDSSYTYYADSVKS  35 9B2 WVRQAPGKGLEWVS  22 SIYSHSMYTYYADSVKS  36 27B3 WVRQAPGKGLEWVS  22 DINNEGGTTYYADSVKG  27 24E3 WVRQAPGKGLEWVS  22 DINNEGGTTYYADSVKG  27 33D8 WVRQAPGKGLEWVS  22 DINNEGGTTYYADSVKG  27 24F2 WVRQAPGKGLEWVS  22 DINNEGGTTYYADSVKG  27 24B6 WVRQAPGKGLEWVS  22 SIYMYDSSTYYADSVKG  29 19G10 WVRQAPGKGLEWVS  22 SIYMYDSSTYYADSVKG  29 45B12 WVRQAPGKGLEWIS  25 DINNEGYETYYADSVKG  37 45D9 WVRQAPGKGLEWIS  25 DINNEGYETYYADSVKG  37 45F8 WVRQAPGKGLEWIS  25 DINNEGYETYYADSVKG  37 45A12 WVRQAPGKGLEWIS  25 DINNEGYETYYADSVKG  37 45B6 WVRQAPGKGLEWIS  25 DINNEGYETYYADSVKG  37 57B6 WVRQAPGKGLEWVS  22 GDNTYDGGTRYQDSVKG 258 59D10 WVRQAPGKGLEWVS  22 GISGVTGGSSTSYADSVKG 259 SEQ SEQ Fab Framework 3 ID CDR3 ID 1C2 RFTISRDHAKNTLILQMNS  38 GSDYEH  49 LKSEDTAVYYCIR 9D1 RFTISRDNAKNTLTLQMN  39 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 8B12 RFTISRDNAKNTLTLQMD  40 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 8C12 RFTISRDNAKNTLTLQMD  40 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 9E1 RFTISTDNAKNTVYLQMN  41 DINRSYGSSWSHFGPIFSS  51 SLKSEDTAVYYCAK 5F4 RFTISRNNAKNTLYLQMN  42 GMGLAEGLTD  52 SLKSEDTAVYYCAR 5B2 RFTISTDNAKNTLYLQMNS  43 SLGLEYGYGD  53 LRSEDSAVYYCTA 6D5 RVTFTADASTSTAYVELSS  44 RRRDFDY  54 LRSEDTAVYYCAS 4D2 RTSISRDTSKNQFTLQLSS  45 VTGEITYNSGSYYYTLNLFD  55 VTPEDTAVYYCAR Y 9A1 RFAISRDNAKNTLYLQMN  46 EGGSGRYWTNEYDY  56 SLKPEDTAVYYCAK 9G2 RFTISTDNAKNTLYLQMNS  47 SSDYEGSFAS  57 LKPDDTAVYYCAG 9B2 RFTISTDNAKNTLYLQMNS 342 SSDYEGLFVS  58 LKPDDTAVYYCAA 27B3 RFTISRDNAKNTLTLQMN  39 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 24E3 RFTISRDNAKNTLTLQMN  39 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 33D8 RFTISRDNAKNTLTLQMN  39 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 24F2 RFTISRDNAKNTLTLQMN  39 DAGYSNHVPIFDS  50 SLKPEDTALYYCVR 24B6 RFTISTDNAKNTVYLQMN  41 DINRSYGSSWSHFGPIFSS  51 SLKSEDTAVYYCAK 19G10 RFTISTDNAKNTVYLQMN  41 DINRSYGSSWSHFGPIFSS  51 SLKSEDTAVYYCAK 45B12 RFTISRDNAKNTLTLQMD  48 DAGYSNHVQIFDS  59 SLKPEDTARYYCVR 45D9 RFTISRDNAKNTLTLQMD  48 DAGYSNHVQIFDS  59 SLKPEDTARYYCVR 45F8 RFTISRDNAKNTLTLQMD  48 DAGYSNHVQIFDS  59 SLKPEDTARYYCVR 45A12 RFTISRDNAKNTLTLQMD  48 DAGYSNHVQIFDS  59 SLKPEDTARYYCVR 45B6 RFTISRDNAKNTLTLQMD  48 DAGYSNHVQIFDS  59 SLKPEDTARYYCVR 57B6 RFTISRDNGKNTLYLQMN 260 DTGRGIMGEYGMDY 262 SLKPEDTAVYYCAK 59D10 RFTISRDNGKNTLYLQMN 261 TSGTYYFIPEYEY 263 SLIPEDTAVYYCAT SEQ Fab FRAMEWORK 4 ID 1C2 WGQGTQVTVSS  60 9D1 WGQGTQVIVAS  61 8B12 WGQGTQVIVAS  61 8C12 WGQGTQVIVAS  61 9E1 WGQGTQVTVSS  60 5F4 WGQGTQVTVSS  60 5B2 WGQGTQVTVSS  60 6D5 WGQGTQVTVSS  60 4D2 WGQGTQVTVSS  60 9A1 WGQGTQVTVSS  60 9G2 WGQGTQVTVSS  60 9B2 WGQGTQVTVSS  60 27B3 WGQGTQVIVAS  61 24E3 WGQGTQVIVAS  61 33D8 WGQGTQVIVAS  61 24F2 WGQGTQVIVAS  61 24B6 WGQGTQVTVSS  60 19G10 WGQGTQVTVSS  60 45B12 WGQGTQVIVAS  61 45D9 WGQGTQVIVAS  61 45F8 WGQGTQVIVAS  61 45A12 WGQGTQVIVAS  61 45B6 WGQGTQVIVAS  61 57B6 WGKGTLVTVSS 264 59D10 WGQGTQVTVSS  60

TABLE 7 sequence of CD70 specific llama-derived VL (Q  in bold encoded by amber STOP codon) SEQ SEQ Fab Framework 1 ID CDR1 ID 1C2 QTVVTQEPSLSVSPGGTVTLTC  62 GLSSGSVTTTNYPG  77 9D1 QAVVTQEPSLSVSPGGTVTLTC  63 GLSSGSVTSSHYPG  78 8B12 QTVVTQEPSLSVSPGGTVTLTC  62 GLSSGSVTSSNYPG  79 8C12 QAVVTQEPSLSVSPGGTVTLTC  63 GLTSGSVTSSNYPD  80 9E1 QAVVTQEPSLSVSPGGTVTLTC  63 GLTSGSVTSSNYPD  80 5F4 QAVVTHPPSLSASPGSSVRLTC  64 TLISGDNIGGYDIS  81 5B2 QSALTQPPSVSGTLGKTLTISC  65 AGTSSDVGYGNYVS  82 6D5 QSALTQPSAVSVSLGQTARITC  66 QGGNARFSSFA  83 4D2 QSVLTQPPSLSASPGSSVRLTC  67 TLSSGNSVGNYDIS  84 9A1 QSALTQPSALSVTLGQTAKITC  68 QGGRLGSSYAH  85 9G2 QSVVTQPPSLSASPGSSVRLTC  69 TLSSGNSVGNYDIS  84 9B2 QAVLTQPPSLSASPGSSVRLTC  70 TLNSANSVGSYDIS  86 27B3 QAVVTQEPSLTVSPGGTVTLTC  71 GLKSGSVTSTNFPT  87 24E3 QAVVTQEPSLSVSPGGTVTLTC  63 GLTSGSVTSDNFPV  88 33D8 QSALTQPSTVSVSLGQTARITC  72 RGDSLERYGTN  89 24F2 QSALTQPSAVSVSLGQTARITC  66 RGDTLRNYHAN  90 24B6 QPVLTQPSAVSVSLGQTARITC  73 QGGYYTH  91 19G10 NFMLTQPSAVSVSLGQTARITC  74 QGGYYTH  91 45B12 QAVLTQPSSVSVSLGQTAKITC  75 QGGNLGLYGAN  92 45D9 QAVLTQPSSVSVSLGQTAKITC  75 QGGNLWLYGAN  93 45F8 QAVLTQPSSVSVSLGQTANITC  76 QGGNLGLYGAN  92 45A12 QAVVTQEPSLSVSPGGTVTLTC  63 GLSSGSATSGNYPE  94 45B6 QAVVTQEPSLSVSPGGTVTLTC  63 GLSSGSVTSSNYPD  95 57B6 QTVVTQEPSLSVSPGGTVTLTC 265 GLKSGSVTSSNYPA 267 59D10 QSVLTQPPSVSGSPGKTVTISC 266 AGTSSDVGYGYYVS 268 SEQ SEQ Fab Framework 2 ID CDR2 ID 1C2 WFQQTPGQAPRTLIY  96 STSSRHS 109 9D1 WYQQTPGQAPRLLIF  97 NTNSRHS 110 8B12 WYQQTPGQAPRVLIY  98 NTNNRHS 111 8C12 WYQQTPGQAPRLLIY  99 NTNSRHS 110 9E1 WYQQTPGQAPRLLIY  99 NTNSRHS 110 5F4 WYQQKTGSPPRYLLY 100 YYSDSYKHQSS 112 5B2 WYQQLPGTAPKLLIY 101 RVSTRAS 113 6D5 WYQQKPGQAPVQVIY 102 YNTNRPS 114 4D2 WYQQKAGSPPRYLLY 103 YYSDSYKNQGS 115 9A1 WYQQKPGQAPVLVIY 104 GNNYRPS 116 9G2 WYQQKAGSPPRYLLY 103 YYSDSVKHQGS 117 9B2 WYQQKAGSPPRYLLY 103 YYSDSLSHQGS 118 27B3 WYQQTPGQAPRLLIY  99 NTNTRHS 119 24E3 WYQQTPGQAPRLLIY  99 TINSRHS 120 33D8 WYQQKPGQAPVLVIY 104 DDDSRPS 121 24F2 WYRQKPGQAPVLVIY 105 GDDIRPS 122 24B6 WYQQKPGQAPVLVIY 104 INNNRPS 123 19G10 WYQQKPGQAPVLVIY 104 VNNNRPS 124 45B12 WYQQNPGRAPILLIY 106 GDNYRPL 125 45D9 WYQQNPGRAPILLIY 106 GDNQRPL 126 45F8 WYQQNPGRAPILLFY 107 GDNYMPL 127 45A12 WYQQTPGQAPRLIIY 108 NTASRHS 128 45B6 WYQQTPGQAPRLLIY  99 NTNSRHS 110 57B6 WYQQTPGQAPRLLIY  99 NTNSRHS 110 59D10 WYQQFPGMAPKLLIY 269 DVNKRAS 270 SEQ SEQ Fab Framework 3 ID CDR3 ID 1C2 GVPSRFSGSISGNKAALTITGAQP 129 ALEEIGSYTYM 148 EDEADYYC 9D1 GVPSRFSGSISGNKAALTITGAQP 129 ALLNIDDGSTM 149 EDEADYYC 8B12 GVPSRYSGSISGNKAALTITGAEP 130 NLHLGSYTPM 150 EDEADYYC 8C12 GVPSRFSGSISGNKAALTITGAQP 131 ALYWGYGTNVDV 151 EDEADYYC 9E1 GVPSRFSGSISGNKAALTITGAQP 129 NLYMGSGGSKV 152 EDEADYYC 5F4 GVPSRFSGSKDASANAGLLLISGL 132 SAYKSGSYKAPV 153 QSEDEADYYC 5B2 GMPDRFSGSKSGNTASLTISGLQ 133 ASYTTNNKPV 154 SEDEADYYC 6D5 GIPARFSGSSSGGAATLTISGAQA 134 QSYESGNYV 155 EDEADYYC 4D2 GVPSRFSGSKDPSANAGLLLISGL 135 SVSNSGTYKPV 156 QAEDEADYYC 9A1 GIPERFSGSSSGDTATLTISGAQA 136 QSGSSNTNVM 157 EDEAVYYC 9G2 GVPSRFSGSSDASANAGLLLISGL 137 SAYKSGSHV 158 QPEDEADYYC 9B2 GVPSRFSGSTDASANAGLLLISGL 138 SAYNRGSHV 159 QPEDEADYYC 27B3 GVPSRFSGSISENKAALTITGAQP 139 ALFISNPSVE 160 EDEAEYFC 24E3 GVPSRFSGSITGNKAILTITGAQPE 140 ALYLENFANE 161 DEADYYC 33D8 GIPERFSGSSSGATAALTISGAQA 141 QSADSSGNAV 162 EDEGDYYC 24F2 GIPERFSGSRLGGTATLTVSGAQA 142 QSSDSSGYRVV 163 EDEADYYC 24B6 GIPERFSGSISGNTATLTISGAQVE 143 QSGSSSTIPV 164 DEADYYC 19G10 GIPERFSGSSSGNTATLTISGAQA 144 QSGSSSTIPV 164 EDEAAYYC 45B12 GIPERFTISKSGGTATLTIDGAQAE 145 QSADYSGNSV 165 DESDYYC 45D9 GIPERFTISKSGGTATLTIDGAQAE 145 QSADYSGNSV 165 DESDYYC 45F8 GIPERFTISKSGGTATLTIDGAQAE 146 QSSDYPGNSV 166 NESDYYC 45A12 GVPGRFSGSISGNKAALTITGAQP 147 LLYMGGSDFNFV 167 EDEADYYC 45B6 GVPSRFSGSISGNKAALTITGAQP 129 ALYMGSGSNNVV 168 EDEADYYC 57B6 GVPSRFSGSISGNKAALTITGAQP 129 ALYMGSGSANAM 271 EDEADYYC 59D10 GIADRFSGSKAGNTASLTISGLQS 272 ASYRSSANAV 273 EDEADYYC SEQ Fab Framework 4 ID 1C2 FGGGTHLTVLG 169 9D1 FGGGTHLTVLG 169 8B12 FGGGTKLTVLG 170 8C12 FGGGTKLTVLG 170 9E1 FGGGTKLTVLG 170 5F4 FGGGTHLTVLG 169 5B2 FGGGTHLTVLG 169 6D5 FGGGTTLTVLG 171 4D2 FGGGSKLTVLG 172 9A1 FGGGTHLTVLS 173 9G2 FGGGTKLTVLG 170 9B2 FGGGTKLTVLG 170 27B3 FGGGTQLTVLS 174 24E3 FGGGTRLTVLG 175 33D8 FGGGTHLTVLG 169 24F2 FGGGTKLTVLG 170 24B6 FGGGTKLTVLG 170 19G10 FGGGTKLTVLG 170 45B12 FGGGTKLTVLG 170 45D9 FGGGTKLTVLG 170 45F8 FGGGTKLTVLG 170 45A12 FGGGTKLTVLG 170 45B6 FGGGTELTVLG 176 57B6 FGGGTHLTVLG 169 59D10 FGGGTHLTVLG 169

TABLE 8 Full Length llama-derived VH Fab Full Length Sequence of VH domain SEQ ID 1C2 ELQVVESGGGLVQPGGSLRLSCAASGFTLSNYWMHWVRQAPRKG 177 LEWVSTISTDNSRTYYADSVKGRFTISRDHAKNTLILQMNSLKSEDT AVYYCIRGSDYEHWGQGTQVTVSS 9D1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178 LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 8B12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 179 LEWVSDINNEGDTTYYADSVKGRFTISRDNAKNTLTLQMDSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 8C12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 179 LEWVSDINNEGDTTYYADSVKGRFTISRDNAKNTLTLQMDSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 9E1 EVQLQESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG 180 LEWVSSIYMYDSSTYYADSVKGRFTISTDNAKNTVYLQMNSLKSED TAVYYCAKDINRSYGSSWSHFGPIFSSWGQGTQVTVSS 5F4 EVQVQESGGGLVHPGGSLKLSCAASGFTFDTYAMSWVRQAPGKG 181 LEWVSAISWSGGETFYAESMKGRFTISRNNAKNTLYLQMNSLKSED TAVYYCARGMGLAEGLTDWGQGTQVTVSS 5B2 QVQLVESGGDLVQPGGSLRLSCAASGFTVSNPAMSWVRQAPGKG 182 LEWVSEITNYGYNRYYADSVKGRFTISTDNAKNTLYLQMNSLRSED SAVYYCTASLGLEYGYGDWGQGTQVTVSS 6D5 EVQLVQPGAELRKPGASVKVSCKASGYTFTSYYIDWVRQAPGQGL 183 EWMGRIDPEDGGTKYAQKFQGRVTFTADASTSTAYVELSSLRSED TAVYYCASRRRDFDYWGQGTQVTVSS 4D2 QVQLQESGPGLVKPSQTLSLACTVSGGSITTSYYYWSWIRQPPGK 184 GLEWMGAIGSRGSTYYSPSLKTRTSISRDTSKNQFTLQLSSVTPED TAVYYCARVTGEITYNSGSYYYTLNLFDYWGQGTQVTVSS 9A1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKG 185 LEWVSDINSGGGSTKYNDSVKGRFAISRDNAKNTLYLQMNSLKPED TAVYYCAKEGGSGRYWTNEYDYWGQGTQVTVSS 9G2 QVQLVESGGGLMQPGGSLRLSCAASGFTFSSSAMSWVRQAPGKG 186 LEWVSSIYSDSSYTYYADSVKSRFTISTDNAKNTLYLQMNSLKPDDT AVYYCAGSSDYEGSFASWGQGTQVTVSS 9B2 QLQVVESGGGLMQPGGSLRLSCAASGFTFSGSAMSWVRQAPGK 187 GLEWVSSIYSHSMYTYYADSVKSRFTISTDNAKNTLYLQMNSLKPD DTAVYYCAASSDYEGLFVSWGQGTQVTVSS 27B3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178 LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 24E3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178 LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 33D8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178 LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 24F2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKG 178 LEWVSDINNEGGTTYYADSVKGRFTISRDNAKNTLTLQMNSLKPED TALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 24B6 EVQLQESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG 180 LEWVSSIYMYDSSTYYADSVKGRFTISTDNAKNTVYLQMNSLKSED TAVYYCAKDINRSYGSSWSHFGPIFSSWGQGTQVTVSS 19G10 EVQLQESGGGVVQPGGSLRLSCAASGFTFDDYAMSWVRQAPGKG 180 LEWVSSIYMYDSSTYYADSVKGRFTISTDNAKNTVYLQMNSLKSED TAVYYCAKDINRSYGSSWSHFGPIFSSWGQGTQVTVSS 45B12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188 LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDT ARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45D9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188 LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDT ARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45F8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188 LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDT ARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45A12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188 LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDT ARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 45B6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSAYYMNWVRQAPGKG 188 LEWISDINNEGYETYYADSVKGRFTISRDNAKNTLTLQMDSLKPEDT ARYYCVRDAGYSNHVQIFDSWGQGTQVIVAS 57B6 QLQLVESGGGLVQPGGSLRLSCAASGFSFSHYAMSWVRQAPGKG 274 LEWVSGDNTYDGGTRYQDSVKGRFTISRDNGKNTLYLQMNSLKPE DTAVYYCAKDTGRGIMGEYGMDYWGKGTLVTVSS 59D10 EVQLVESGGGLVQPGGSLRLSCAASELSFSISEMTWVRQAPGKGL 275 EWVSGISGVTGGSSTSYADSVKGRFTISRDNDKNTLYLQMNSLIPE DTAVYYCATTSGTYYFIPEYEYWGQGTQVTVSS

TABLE 9 Full Length llama-derived VL (Q in bold encoded by amber STOP codon) Fab Full Length Sequence SEQ ID 1C2 QTVVTQEPSLSVSPGGTVTLTCGLSSGSVTTTNYPGWFQQTPGQA 189 PRTLIYSTSSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL EEIGSYTYMFGGGTHLTVLG 9D1 QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSHYPGWYQQTPGQ 190 APRLLIFNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCA LLNIDDGSTMFGGGTHLTVLG 8B12 QTVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSNYPGWYQQTPGQA 191 PRVLIYNTNNRHSGVPSRYSGSISGNKAALTITGAEPEDEADYYCNL HLGSYTPMFGGGTKLTVLG 8C12 QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSSNYPDWYQQTPGQA 192 PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL YWGYGTNVDVFGGGTKLTVLG 9E1 QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSSNYPDWYQQTPGQA 193 PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCNL YMGSGGSKVFGGGTKLTVLG 5F4 QAVVTHPPSLSASPGSSVRLTCTLISGDNIGGYDISWYQQKTGSPP 194 RYLLYYYSDSYKHQSSGVPSRFSGSKDASANAGLLLISGLQSEDEA DYYCSAYKSGSYKAPVFGGGTHLTVLG 5B2 QSALTQPPSVSGTLGKILTISCAGTSSDVGYGNYVSWYQQLPGTA 195 PKLLIYRVSTRASGMPDRFSGSKSGNTASLTISGLQSEDEADYYCA SYTTNNKPVFGGGTHLTVLG 6D5 QSALTQPSAVSVSLGQTARITCQGGNARFSSFAWYQQKPGQAPV 196 QVIYYNTNRPSGIPARFSGSSSGGAATLTISGAQAEDEADYYCQSY ESGNYVFGGGTTLTVLG 4D2 QSVLTQPPSLSASPGSSVRLTCTLSSGNSVGNYDISWYQQKAGSP 197 PRYLLYYYSDSYKNQGSGVPSRFSGSKDPSANAGLLLISGLQAEDE ADYYCSVSNSGTYKPVFGGGSKLTVLG 9A1 QSALTQPSALSVTLGQTAKITCQGGRLGSSYAHWYQQKPGQAPVL 198 VIYGNNYRPSGIPERFSGSSSGDTATLTISGAQAEDEAVYYCQSGS SNTNVMFGGGTHLTVLS 9G2 QSVVTQPPSLSASPGSSVRLTCTLSSGNSVGNYDISWYQQKAGSP 199 PRYLLYYYSDSVKHQGSGVPSRFSGSSDASANAGLLLISGLQPEDE ADYYCSAYKSGSHVFGGGTKLTVLG 9B2 QAVLTQPPSLSASPGSSVRLTCTLNSANSVGSYDISWYQQKAGSP 200 PRYLLYYYSDSLSHQGSGVPSRFSGSTDASANAGLLLISGLQPEDE ADYYCSAYNRGSHVFGGGTKLTVLG 27B3 QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQA 201 PRLLIYNTNTRHSGVPSRFSGSISENKAALTITGAQPEDEAEYFCAL FISNPSVEFGGGTQLTVLS 24E3 QAVVTQEPSLSVSPGGTVTLTCGLTSGSVTSDNFPVWYQQTPGQA 202 PRLLIYTINSRHSGVPSRFSGSITGNKAILTITGAQPEDEADYYCALY LENFANEFGGGTRLTVLG 33D8 QSALTQPSTVSVSLGQTARITCRGDSLERYGTNWYQQKPGQAPVL 203 VIYDDDSRPSGIPERFSGSSSGATAALTISGAQAEDEGDYYCQSAD SSGNAVFGGGTHLTVLG 24F2 QSALTQPSAVSVSLGQTARITCRGDTLRNYHANWYRQKPGQAPVL 204 VIYGDDIRPSGIPERFSGSRLGGTATLTVSGAQAEDEADYYCQSSD SSGYRVVFGGGTKLTVLG 24B6 QPVLTQPSAVSVSLGQTARITCQGGYYTHWYQQKPGQAPVLVIYIN 205 NNRPSGIPERFSGSISGNTATLTISGAQVEDEADYYCQSGSSSTIPV FGGGTKLTVLG 19G10 NFMLTQPSAVSVSLGQTARITCQGGYYTHWYQQKPGQAPVLVIYV 206 NNNRPSGIPERFSGSSSGNTATLTISGAQAEDEAAYYCQSGSSSTI PVFGGGTKLTVLG 45B12 QAVLTQPSSVSVSLGQTAKITCQGGNLGLYGANWYQQNPGRAPIL 207 LIYGDNYRPLGIPERFTISKSGGTATLTIDGAQAEDESDYYCQSADY SGNSVFGGGTKLTVLG 45D9 QAVLTQPSSVSVSLGQTAKITCQGGNLWLYGANWYQQNPGRAPIL 208 LIYGDNQRPLGIPERFTISKSGGTATLTIDGAQAEDESDYYCQSADY SGNSVFGGGTKLTVLG 45F8 QAVLTQPSSVSVSLGQTANITCQGGNLGLYGANWYQQNPGRAPIL 209 LFYGDNYMPLGIPERFTISKSGGTATLTIDGAQAENESDYYCQSSDY PGNSVFGGGTKLTVLG 45A12 QAVVTQEPSLSVSPGGTVTLTCGLSSGSATSGNYPEWYQQTPGQ 210 APRLIIYNTASRHSGVPGRFSGSISGNKAALTITGAQPEDEADYYCL LYMGGSDFNFVFGGGTKLTVLG 45B6 QAVVTQEPSLSVSPGGTVTLTCGLSSGSVTSSNYPDWYQQTPGQA 211 PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL YMGSGSNNVVFGGGTELTVLG 57B6 QTVVTQEPSLSVSPGGTVTLTCGLKSGSVTSSNYPAWYQQTPGQA 276 PRLLIYNTNSRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCAL YMGSGSANAMFGGGTHLTVLG 59D10 QSVLTQPPSVSGSPGKTVTISCAGTSSDVGYGYYVSWYQQFPGMA 277 PKLLIYDVNKRASGIADRFSGSKAGNTASLTISGLQSEDEADYYCAS YRSSANAVFGGGTHLTVLG

Example 13 Germlining of Anti-CD70 mAb 27B3

Human germline genes segments with the same canonical fold structure and the highest amino acid sequence identity to the VH and VL regions of mAb 27B3 were identified by comparison with known human germline gene sequences. The closest human VH and VL regions to mAb 27B3 was human VH3-48 (89.7% FR identity) and human VL8-61 (86.1% FR identity), respectively. The closest human germline JH and JL human germline gene segment sequences were JH5 and JL7, respectively. Sequence alignment of the VH and VL regions of mAb 27B3 with the closest human germline VH and VL sequences are set forth in FIG. 12. Comparison of the V regions of SIMPLE antibody 27B3 and human germline sequences identified 12 candidate humanizing mutations in the VH region and 13 in the VL region (2 in CDR1 and 1 in CDR2). An overview of the humanizing mutations in the VH and VL sequences of the 27B3, together with the needed library sizes to cover the introduced diversity, is set forth in Table 10 and 11.

TABLE 10 Targeted mutations in the 27B3 VH amino acid sequence. Mutations included in library Human Human Human germline Kabat Camelid germline aa germline aa aa Postion aa option 1 option 2 option 3 Probability  46 E V 0.50  74 A S 0.50  77 T S 0.50  79 T Y 0.50 83/84 KP KT RA 0.33  89 L V 0.50 93/94 AR AK VR 0.33 108 Q L T M 0.25 110 I T 0.50 112 A S 0.50 4608 

denotes the library size needed to cover all potential mutants.

TABLE 11 Targeted mutations in the 27B3 VL amino acid sequence. Human Human Human germline germline germline Kabat Camelid aa aa aa Postion aa option 1 option 2 option 3 Probability 2 A T 0.50 11-12 LT FS 0.50 26* K T 0.50 30* T D 0.50 46  L T 0.50 53* T S 0.50 60  S D 0.50 61  R C 0.50 67/68 SE LG 0.50 80/81/84/85 PEDEAE PEDESD ADDESD ADDEAE 0.25 87  F Y 0.50 4096 

denotes the library size needed to cover all potential mutants. *denotes mutations in the CDR1 or CDR2 regions.

Germlined libraries of VH_27B3 and VL_27B3 encompassing all combinations of humanizing mutations were created by PCR based assembly (see e.g., Stemmer et al., Gene (1995) 164: 49-53). Overlapping oligonucleotides with specific mutations on certain positions were assembled by PCR. The library contained both human and llama amino acids at each mutated position to prevent complete loss of binding in the event that the wild type llama residue was critical for high affinity binding (see e.g., Baca et al., J. Biol. Chem. (1997) 272: 10678-10684; Tsurushita et al., J. Immunol. Methods (2004) 295: 9-19). The VH library contained about 1×10¹⁰ clones and the VL library about 8×10⁹ clones. The VH and VL libraries were combined and reformatted into a single Fab library with a size of 1×10¹⁰ clones (91% with full length Fab insert) suitable for phage display screening. To required diversity to cover all possible mutations as mentioned in tables 10 and 11 was exceeded in the primary heavy chain and light chain libraries, but also the combined Fab library was large enough to cover all possible permutations (1.89×10⁷).

Example 14 Selection of Germlined 27B3 Fabs with High Human FR Identity

Phage display was used to select for germlined 27B3 Fabs with both a high affinity for CD70 and high human FR identity. Specifically, Flag-TNC-CD70 was biotinylated and was incubated at various concentrations with different amounts of Fab expressing phage. Complexes of phage and Flag-TNC-CD70 were then captured on a streptavidin-coated microtiter plate and washed with non-biotinylated Flag-TNC-CD70 at 37° C. Phage were eluted with Trypsin and used for infection of TG1 cells. Five rounds of selection were performed, with the wash stringency increased in each round. Details of the conditions used for each round of selection are set forth in Table 12.

TABLE 12 Conditions for selection of germlined 27B3 Fabs Phage input Antigen Washing Wash Round (μl) [pM] time temperature Washing antigen 1 10 24000-2400-240 1 hour RT PBS 2 1 2400-240-24 ON 37° C. 24 nM CD70 3 0.1 240-24-2.4 ON 37° C. 2.4 nM CD70 4 0.01 24-2.4-0.24 3 days 37° C. 240 pM CD70 5 0.1 2.4-0.24-0.024 6 days 37° C. 24 pM CD70

Phage eluted from rounds 3, 4 and 5 of selection were transfected into TG1 and plated clonally; individual clones were picked randomly for further analysis. Periplasmic fractions containing Fabs were prepared from IPTG induced small scale cultures the off rate of each Fab was determined using a CD70 coated CM5 chip in a Biacore binding assay. Clones with Fab having off rates similar to that of 27B3 were sequenced. Off rates for clones with a total human FR sequence identity above 94% are set forth in Table 13, along with the percentage human FR identity of the individual clone (VH, VL and total).

The complete amino acid sequences of the sequenced Fab clones are set forth in tables 14 and 15 and CDR amino acid sequence variants of 27B3 are set forth in table 16. Inspection of these sequences reveals that framework 1 of the light chain often has extra (non human) mutations in the primer region at amino acid 2 (A instead of T). Variants of clones 35G3, 40F1 and 39C3 (clones 53C1, 53B1 and 53E1, respectively) were made that have the corresponding human amino acid at position 2. Moreover, clone 41D12 was further germlined by combining the heavy chain of 41D12 with the light chain of 40F1 (53A2) or with the light chain of 53B1 (53H1).

TABLE 13 Off rate of germlined 27B3 Fab clones with more than 94% total human FR identity. % FR % FR Total % Fab identity for identity for FR Isolation Off rate Clone VL VH identity frequency [10⁻⁴s⁻¹] 27B3 86.0 89.5 87.9 1.4 36A9 97.5 92.0 94.5 1 1.3 53F1 97.5 92.0 94.5 1 2.3 36D6 94.9 94.3 94.6 2 1.4 53G1 96.2 94.0 95.0 1 2.0 35G3 94.9 95.4 95.2 1 1.7 35F6 94.9 95.4 95.2 1 ND 36G2 93.6 96.6 95.2 7 1.1 39D5 93.7 96.6 95.2 2 1.2 42D12 93.7 96.6 95.2 2 1.6 35G1 97.5 94.3 95.7 1 2.3 41D12 92.4 98.9 95.9 4 2.3 41H8 94.9 97.0 96.0 1 2.2 35G2 96.2 96.6 96.4 1 1.7 40F1 94.9 98.0 96.6 1 1.6 39C3 97.5 97.7 97.6 6 2.5

TABLE 14 VH amino acid sequences of germlined 27B3 clones with more than 94% total human identity, with sequence identifiers broken down by FR and CDR. Humanizing mutations are italicized and primer-introduced mutations are bolded. HEAVY CHAIN CLONE FRAMEWORK 1 CDR1 FRAMEWORK 2 27B3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 36A9 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 36D6 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53G1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ 12 NO: 2 SEQ 12 NO: 11 SEQ ID NO: 22 35G3 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53C1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 35F6 EVQLVESGGGLVQPGGSLRLSCASSGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 274 SEQ ID NO: 11 SEQ ID NO: 22 36G2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 39D5 EVQLVESGGGLVQPGGSLRLSCAASGFTFS GYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 248 SEQ ID NO: 22 42D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 35G1 EVQLVESGGGLVQPGGSVRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 41D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 41H8 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ 12 NO: 2 SEQ 12 NO: 11 SEQ ID NO: 22 35G2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 40F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53B1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 39C3 EVQLVASGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 275 SEQ ID NO: 11 SEQ ID NO: 22 53E1 EVQLVASGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 275 SEQ ID NO: 11 SEQ ID NO: 22 53H1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 53A2 EVQLVESGGGLVQPGGSLRLSCAASGFTFS VYYMN WVRQAPGKGLEWVS SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 22 HEAVY CHAIN CLONE CDR2 FRAMEWORK 3 CDR3 FRAMEWORK 4 27B3 DINNEGGTTYYADSVKG RFTISRDNAKNTLTLQMNSLKPEDTALYYCVR DAGYSNHVPIFDS WGQGTQVIVAS SEQ ID NO: 27 SEQ ID NO: 39 SEQ ID NO: 50 SEQ ID NO: 61 36A9 DINNEGGTTYYADSVKG RFTISRDNSKNTLTLQMNSLKPEDTAVYYCVR DAGYSNHVPIFDS WGQGTQVIVSS SEQ ID NO: 27 SEQ ID NO: 350 SEQ ID NO: 50 SEQ ID NO: 287 53F1 DINNEGGTTYYADSVKG RFTISRDNAKNTLTLQMNSLKPEDTAVYYCVR DAGYSNHVPIFDS WGQGTQVTVAS SEQ ID NO: 27 SEQ ID NO: 351 SEQ 12 NO: 50 SEQ ID NO: 288 36D6 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTALYYCVR DAGYSNHVPIFDS WGQGTTVTVSS SEQ ID NO: 27 SEQ ID NO: 278 SEQ ID NO: 50 SEQ ID NO: 289 53G1 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVR DAGYSNHVPIFDS WGQGTTVIVSS SEQ ID NO: 27 SEQ ID NO: 279 SEQ ID NO: 50 SEQ ID NO: 290 35G3 DINNEGGTTYYADSVKG RFTISRDNSKNSLTLQMNSLKPEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 280 SEQ ID NO: 50 SEQ ID NO: 293 53C1 DINNEGGTTYYADSVKG RFTISRDNSKNSLTLQMNSLKPEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 280 SEQ ID NO: 50 SEQ ID NO: 293 35F6 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR DAGYSNHVPIFDS WGQGTTVTVAS SEQ ID NO: 27 SEQ ID NO: 281 SEQ ID NO: 50 SEQ ID NO: 291 36G2 DINNEGGTTYYADSVKG RFTISRDNAKNSLTLQMNSLRAEDTAVYYCVR DAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 27 SEQ ID NO: 282 SEQ ID NO: 50 SEQ ID NO: 292 39D5 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCVR DAGYSNHVPIFDS WGQGTTVTVAS SEQ ID NO: 27 SEQ ID NO: 283 SEQ ID NO: 50 SEQ ID NO: 291 42D12 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVR DAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 27 SEQ ID NO: 281 SEQ ID NO: 50 SEQ ID NO: 292 35G1 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVAS SEQ ID NO: 27 SEQ ID NO: 284 SEQ ID NO: 50 SEQ ID NO: 294 41D12 DINNEGGTTYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 285 SEQ ID NO: 50 SEQ ID NO: 293 41H8 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTQVTVAS SEQ ID NO: 27 SEQ ID NO: 286 SEQ ID NO: 50 SEQ ID NO: 288 35G2 DINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 284 SEQ ID NO: 50 SEQ ID NO: 293 40F1 DINNEGGATYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 249 SEQ ID NO: 285 SEQ ID NO: 50 SEQ ID NO: 292 53B1 DINNEGGATYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTQVTVSS SEQ ID NO: 249 SEQ ID NO: 285 SEQ ID NO: 50 SEQ ID NO: 292 39C3 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTTVTVSS SEQ ID NO: 27 SEQ ID NO: 286 SEQ ID NO: 50 SEQ ID NO: 289 53E1 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTTVTVSS SEQ ID NO: 27 SEQ ID NO: 286 SEQ ID NO: 50 SEQ ID NO: 289 53H1 DINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 285 SEQ ID NO: 50 SEQ ID NO: 293 53A2 DINNEGGTTYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCAR DAGYSNHVPIFDS WGQGTLVTVSS SEQ ID NO: 27 SEQ ID NO: 285 SEQ ID NO: 50 SEQ ID NO: 293 VH amino acid sequences of germlined 27B3 clones with more than 94% total human identity, with sequence identifiers for the full VH amino acid sequence. Humanizing mutations are italicized and primer-introduced mutations are bolded. HEAVY SEQ ID CHAIN NO: CLONE FULL VH SEQUENCE 178 27B3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLTLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTQVIVAS 212 36A9 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNTLTLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVIVSS 213 53F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLTLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVAS 214 36D6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTALYYCVRDAGYSNHVPIFDSWGQGTTVTVSS 215 53G1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVRDAGYSNHVPIFDSWGQGTTVIVSS 216 35G3 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNSLTLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 217 53C1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNSLTLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 218 35F6 EVQLVESGGGLVQPGGSLRLSCASSGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTTVTVAS 219 36G2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNSLTLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVSS 220 39D5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLTLYMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTTVTVAS 221 42D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVRDAGYSNHVPIFDSWGQGTQVTVSS 222 35G1 EVQLVESGGGLVQPGGSVRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVAS 223 41D12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 224 41H8 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTQVTVAS 225 35G2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 226 40F1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGATYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTQVTVSS 227 53B1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGATYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTQVTVSS 228 39C3 EVQLVASGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RETISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTTVTVSS 229 53E1 EVQLVASGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTTVTVSS 223 53H1 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS 223 53A2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSVYYMNWVRQAPGKGLEWVSDINNEGGTTYYADSVKG RFTISRDNSKNSLYLQMNSLRAEDTAVYYCARDAGYSNHVPIFDSWGQGTLVTVSS

TABLE 15 VL amino acid sequences of germlined 27B3 clones with more than 94% total human identity, with sequence identifiers broken down by FR and CDR. Humanizing mutations are italicized and primer-introduced mutations are bolded. LIGHT CHAIN CLONE FRAMEWORK 1 CDR1 FRAMEWORK 2 27B3 QAVVTQEPSLTVSPGGTVTLTC GLKSGSVTSTNFPT WYQQTPGQAPALLIY SEQ ID NO: 71 SEQ ID NO: 87 SEQ ID NO: 99 36A9 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 53F1 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNEPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 36D6 QTVVTQEPSLTVSPGGTVTLTC GLKSGSVTSTNFPT WYQQTPGQAPALLIY SEQ ID NO: 296 SEQ ID NO: 87 SEQ ID NO: 99 53G1 QTVVTQEPSFSVSPGGTVTLTC GLTSGSVTSTNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 251 SEQ ID NO: 99 35G3 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 53C1 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 35F6 QTVVTQEPSFSVSPGGTVTLTC GLTSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 252 SEQ ID NO: 99 36G2 QAVVTQEPSLTVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 71 SEQ ID NO: 250 SEQ ID NO: 99 39D5 QTVVTQEPSLTVSPGGTVTLTC GV TSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 296 SEQ ID NO: 253 SEQ ID NO: 99 42D12 QAVVTQEPSFSVSPGGTVTLTC GLTSGSVTSTNFPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 251 SEQ ID NO: 99 35G1 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 41D12 QAVVTQEPSLTVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 71 SEQ ID NO: 250 SEQ ID NO: 99 41H8 QAVVTQEPSFSVSPGGTVTLTC GLTSGSVTSTNFPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 251 SEQ ID NO: 99 35G2 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSTNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 87 SEQ ID NO: 99 40F1 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 53B1 QTVVTQFPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 39C3 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 53E1 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 53H1 QTVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 294 SEQ ID NO: 250 SEQ ID NO: 99 53A2 QAVVTQEPSFSVSPGGTVTLTC GLKSGSVTSDNFPT WYQQTPGQAPALLIY SEQ ID NO: 295 SEQ ID NO: 250 SEQ ID NO: 99 LIGHT CHAIN CLONE CDR2 FRAMEWORK 3 CDR3 FRAMEWORK 4 27B3 NTNTRHS GVPSRFSGSISENKAALTITGAQPEDEAEYFC ALFISNPSVE FGGGTQLTVLS SEQ ID NO: 119 SEQ ID NO: 139 SEQ ID NO: 160 SEQ ID NO: 174 36A9 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 53F1 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 36D6 NTNSRHS GVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 53G1 NTNSRHS GVPSRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 298 SEQ ID NO: 160 SEQ ID NO: 309 35G3 NTNSRHS GVPDRFSGSILGNKAALTIRGAQADDEAEYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 299 SEQ ID NO: 160 SEQ ID NO: 309 53C1 NTNSRHS GVPDRFSGSILGNKAALTIRGAQADDEAEYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 299 SEQ 10 NO: 160 SEQ ID NO: 309 35F6 NTNSRHS GVPDRFSGSILGNKAALTITGAQPEDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 300 SEQ ID NO: 160 SEQ ID NO: 309 36G2 NTNTRHS GVPDRFSGSILGNKAALTITGAQPEDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 301 SEQ ID NO: 160 SEQ ID NO: 309 39D5 NTNTRHS GVPDRFSGSILGNKAALTITGAQPEDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 301 SEQ ID NO: 160 SEQ ID NO: 309 42D12 NTNTRHS GVPSRFSGSILGNKAALTITGAQADEDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 302 SEQ ID NO: 160 SEQ ID NO: 309 35G1 NTNSRHS GVPDRFSGSILGNKAALTITGAQADDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 297 SEQ ID NO: 160 SEQ ID NO: 309 41D12 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 303 SEQ ID N0: 160 SEQ ID NO: 309 41H8 NTNSRHS GVPDRFSGSILGNKAALTITGAQPEDESDYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 300 SEQ ID NO: 160 SEQ ID NO: 309 35G2 NTNSRHS GVPDRFSGSILGNKAALTITGAQADDEADYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 110 SEQ ID NO: 304 SEQ 10 NO: 160 SEQ ID NO: 309 40F1 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 53B1 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 39C3 NTNTRHS GVPSRFSGSILGNKAALTITGAQADDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 305 SEQ ID NO: 160 SEQ ID NO: 309 53E1 NTNTRHS GVPSRFSGSILGNKAALTITGAQADDESDYYC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 305 SEQ ID NO: 160 SEQ ID NO: 309 53H1 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 53A2 NTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFC ALFISNPSVE FGGGTQLTVLG SEQ ID NO: 119 SEQ ID NO: 303 SEQ ID NO: 160 SEQ ID NO: 309 VL amino acid sequences of germlined 27B3 clones with more than 94% total human identity, with sequence identifiers for the full VL sequence. Humanizing mutations are italicized and primer-introduced mutations are bolded. LIGHT SEQ ID CHAIN NO: CLONE FULL VL SEQUENCE 201 27B3 QAVVTQEPSITVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQAPRLLIYNTNTRHS GVPSRFSGSISENKAALTITGAQPEDEAEYFCALFISNPSVEFGGGTQLTVLS 230 36A9 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 231 53F1 QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 232 36D6 QTVVTQEPSLTVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHS GVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 233 53G1 QTVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHS GVPSRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 234 35G3 QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHS GVPDRFSGSILGNKAALTIRGAQADDEAEYYCALFISNPSVEFGGGTQLTVLG 235 53C1 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPALLIYNTNSRHS GVPDRFSGSILGNKAALTIRGAQADDEAEYYCALFISNPSVEFGGGTQLTVLG 236 35F6 QTVVTQEPSFSVSPGGTVILTCGLTSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHS GVPDRFSGSILGNKAALTITGAQPEDESDYFCALFISNPSVEFGGGTQLTVLG 237 36G2 QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQPEDESDYYCALFISNPSVEFGGGTQLTVLG 238 39D5 QTVVTQEPSLTVSPGGTVTLTCGV TKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQPEDESDYYCALFISNPSVEFGGGTQLTVLG 239 42D12 QAVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNFPTWYQQTPGQAPRLLIYNTNTRHS GVPSRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 240 35G1 QTVVTQEPSFSVSPGGTVTLYCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNSRHS GVPDRFSGSILGNKAALTITGAQADDESDYFCALFISNPSVEFGGGTQLTVLG 241 41D12 QAVVTQEPSLTVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPALLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 242 41H8 QAVVTQEPSFSVSPGGTVTLTCGLTSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHS GVPDRFSGSILGNKAALTITGAQPEDESDYFCALFISNPSVEFGGGTQLTVLG 243 35G2 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSTNFPTWYQQTPGQAPRLLIYNTNSRHS GVPDRFSGSILGNKAALTITGAQADDEADYFCALFISNPSVEFGGGTQLTVLG 244 40F1 QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 245 53B1 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 246 39C3 QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPSRFSGSTLGNKAALTITGAQADDESDYYCALFISNPSVEFGGGTQLTVLG 247 53E1 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPSRFSGSILGNKAALTITGAQADDESDYYCALFISNPSVEFGGGTQLTVLG 244 53H1 QTVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG 245 53A2 QAVVTQEPSFSVSPGGTVTLTCGLKSGSVTSDNFPTWYQQTPGQAPRLLIYNTNTRHS GVPDRFSGSILGNKAALTITGAQADDEAEYFCALFISNPSVEFGGGTQLTVLG

Example 15 Temperature Stability of Germlined 27B3 mAbs

Several germlined 27B3 mAb variants were expressed in HEK293 cells as full-length human IgG1 molecules. After protein A purification, the temperature stability of the germlined 27B3 mAb clones was assessed. Specifically, each mAb was incubated at various temperatures (2 or 5° C. intervals apart) for 1 hour at a concentration of 100 μg/ml in PBS buffer. The temperature of the mAbs was then decreased in a controlled manner (reduction to 25° C. over 2 hours, followed by overnight incubation at 4° C.) and centrifugation was used to remove aggregates. The amount of active mAb (as percentage of the total mAb concentration) in the supernatant was measured by Biacore.

The results (set forth in Table 17) demonstrate that most clones have a similar melting temperature (T_(m)) as wild type 27B3 has. Surprisingly, clones 41D12 and 35G2 show to have an increased thermostability as compared to 27B3.

TABLE 17 Temperature stability of germlined 27B3 mAbs mAb Clone T_(m) [° C.] 27B3 (WT) 64.7 64.6 35G1 63.7 35G3 61.7 53A2 62.2 62.2 41D12 65.9 65.9 35G2 65.6 66.0 40F1 63.5 53H1 64.1

The temperature stability of germlined 27B3 mAbs: 41D12, 35G2 and 40F1 was further studied using a variety of techniques. Aliquots (1 ml) of the test mAbs at a concentration of 100 μg/ml were prepared in Delbucco's PBS containing 0.02% Tween. A negative control aliquot consisting of buffer only (Delbucco's PBS containing 0.02% Tween) and a positive control aliquot consisting of mAb in Delbucco's PBS containing 0.02% Tween were also prepared. For each mAb preparation, aliquots were stored at 4° C., at RT and at 37° C. for a period of 0-8 weeks. 50 μl samples were removed from the aliquots on days 1, 7, 14, 21, 28, 35, 56 and 92 for analysis. A sample was stored at −20° C. and used as reference.

15.1 Gelfiltration Analysis of Samples

Samples taken at each time point were also analysed by gelfiltration using a Superdex200 10/300 GL column. The 50 μl test sample was added to 200 μl PBS+0.02% Tween-20. A sample (125 μl) was taken for gelfiltration analysis. Samples were centrifuged before analysis to remove larger aggregates. The % of monomeric peak and area of monomeric peak was measured, as well as the retention volume. The results are shown in Tables 19, and 21.

TABLE 19 Results of PBS stability study—% monomeric peak d1 d7 d14 d21 d28 d35 d56 d92 35G2 Reference 99.82 99.84 99.81 99.81 99.80 99.87 40F1 99.85 99.83 99.83 99.83 99.87 99.86 41D12 99.84 99.81 99.84 99.75 99.73 99.23 99.82 99.84 35G2  4° C. 99.85 99.75 99.74 99.83 99.66 99.84 40F1 99.65 99.29 99.73 99.77 99.72 41D12 99.88 99.76 99.83 99.8 99.84 99.81 99.49 35G2 RT 99.78 99.83 99.81 99.87 99.83 40F1 99.87 99.83 99.84 99.77 99.77 41D12 99.88 99.86 99.87 99.78 99.73 99.80 99.71 35G2 37° C. 99.86 99.80 99.72 99.68 99.48 99.32 40F1 99.83 99.78 99.78 99.77 99.40 99.27 41D12 99.89 99.77 99.61 99.71 99.45 99.02 98.66 97.46

TABLE 20 Results of PBS stability study—area under curve of the monomeric peak (mAU*ml) d1 d7 d14 d21 d28 d35 d56 d92 35G2 Refer- 67.34 68.46 64.92 66.70 66.69 66.37 40F1 ence 67.04 67.96 70.36 69.62 69.53 67.23 41D12 66.41 67.64 61.17 68.53 68.60 66.08 67.48 67.06 35G2  4° C. 68.41 67.58 62.55 68.64 68.12 68.42 40F1 69.86 67.31 65.55 66.76 68.99 41D12 68.38 69.87 65.61 68.98 69.99 68.85 67.33 35G2 RT 66.76 67.23 71.40 68.55 68.45 40F1 68.42 69.53 69.17 68.02 70.32 41D12 66.47 69.01 64.32 69.72 71.56 69.36 67.16 35G2 37° C. 67.52 67.14 64.49 61.89 66.51 65.64 40F1 66.48 67.69 67.35 68.02 68.61 67.23 41D12 67.09 68.13 61.51 68.97 68.37 68.25 67.78 66.51

TABLE 21 Results of PBS stability study—retention volume (ml) d1 d7 d14 d21 d28 d35 d56 d92 35G2 Refer- 12.17 12.13 12.13 12.15 12.17 12.20 40F1 ence 12.14 12.09 12.11 12.12 12.14 12.16 41D12 12.12 12.08 12.10 12.10 12.12 12.14 12.10 12.13 35G2  4° C. 12.16 12.12 12.14 12.16 12.17 12.20 40F1 12.13 12.09 12.11 12.12 12.14 41D12 12.12 12.08 12.10 12.10 12.13 12.10 12.13 35G2 RT 12.16 12.12 12.14 12.16 12.17 40F1 12.14 12.09 12.10 12.12 12.13 41D12 12.12 12.07 12.10 12.10 12.28 12.09 12.13 35G2 37° C. 12.16 12.12 12.13 12.14 12.16 12.17 40F1 12.13 12.09 12.09 12.12 12.12 12.14 41D12 12.12 12.08 12.08 12.09 12.10 12.12 12.06 12.08

The results of gelfiltration analysis of 35G2, 40F1 and 41D12 samples taken after 5 weeks incubation at 37° C. are shown in FIG. 13. Two minor peaks are visible at a higher retention time (indicating the presence of some aggregates) and lower retention time (indicating the presence of some degradation product). The results demonstrate that the majority of the protein is intact and does not aggregate.

Analysis of Samples by BIACORE

Samples taken at certain time points were tested for potency in CD70 binding using Biacore. The 50 μl sample was added to 200 μl PBS+0.02% Tween, and diluted 1/400 as follows: 5 μl of sample (1 mg/ml)+195 μl HBS-EP+(Biacore buffer), further diluted 10 μl+90 μl HVS-EP+=2.5 μg/ml. Biacore analysis was performed using a highly CD70 coated CM5 chip (4000 RU). The results for mAbs 40F1, 35G2 and 41D12 are shown in FIG. 16. The reference sample is the 100% sample.

The results demonstrate that the mAbs loose some potency (affinity for the antigen CD70) over time when incubated at 37° C. For 41D12, both the slope and the maximal signal (R0) are plotted (see FIGS. 16C and D).

Next, the freeze-thaw stability of germlined 27B3 mAbs: 41D12, 35G2 and 40F1 was studied. Therefore, a 0.7 ml aliquot of the mAbs (at 5 mg/ml) was frozen for at least 6 hours at −20° C. and thawed for 1 hour at RT. This cycle was repeated 9× (so 10 freeze-thaw cycles in total) and samples were analyzed by gelfiltration as above. The results demonstrate that the mAbs are stable upon freeze thawing for 10 cycles: no degradation products or aggregates were observed in gelfiltration.

TABLE 22 Results of gelfiltration stability study PBS (left = value after 10 F/T cycles, right is value for reference sample) 35G2 40F1 41D12 Gel Filtration 10/ref 10/ref 10/ref % monomeric peak 99.9/99.8 99.8/99.7 99.8/99.8 Area monomeric peak 67.5/64.7 65.9/67.2 68.2/67.8 Retention time 12.20/12.21 12.15/12.15 12.12/12.12

Samples were also tested for potency in Biacore as described above. The results demonstrate that after 10 freeze-thaw cycles the mAb still has 97% of its potency (for all three mAbs tested.

TABLE 23 Potency in Biacore upon 10 freeze-thaw cycles (%) 35G2 40F1 41D12 Reference 100 100 100 10xFT 97.35 96.96 97.02

Example 16 ADCC Potency of Germlined 27B3 mAbs

Several germlined 27B3 variants were expressed in HEK293 cells as full-length human IgG1 molecules. After protein A purification, the ADCC potency of the germlined 27B3 mAbs was assessed. ADCC was measured using as described above in example 7.

The relative IC50's for the germlined 27B3 mAbs compared to the parental 27B3 are given in table 18 for both ADCC potency and CD70/CD27 blocking potency in the Raji co-culture assay (as described in example 6). A figure of <1.0 denotes improved IC50 for germlined variant relative to parental 27B3. From these data it can be concluded that germlined variant 41D12 maintained the ADCC potency as well as the CD70/CD27 blocking potency combined with the best T_(m).

TABLE 18 Relative IC50's of germlined 27B3 variants in the 786-O based ADCC assay and neutralization in Raji based bioassay compared to that of parental mAb clone Raji co-culture mAb assay IC50 relative ADCC IC50 of germlined mAb clone to 27B3 relative to 27B3 27B3 1.0 1.0 1.0 1.0 1.0 1.0 1.0 53A2 0.8 1.5 1.9 1.5 4.6 0.7 41D12 1.2 1.7 1.1 2.2 1.4 2.0 2.0 35G2 1.4 1.7 2.1 4.4 1.4 1.9 2.3 40F1 1.8 1.0 1.5 1.3 1.5 2.1 1.6 53H1 1.6 0.9 1.8 3.4 2.1 1.9 1.7

Example 17 Construction and Selection of Cell Lines Expressing the Non-fucosylated CD70 mAb, ARGX-110

A double gene vector encoding the VH and VL amino acid sequences of germlined 27B3 clone 41D12 was produced (za allotype). Nucleotide sequences encoding the heavy chain (SEQ ID NO:344) and light chain (SEQ ID NO:345) of 41D12 are given in Table 30 below.

Potelligent® CHOK1SV cells were stably transfected with the double gene vector by electroporation. Six rounds of transfection were performed. In each round, the cells from each electroporation were added to 200 mL of chemically defined animal component free (CDACF) medium CM63 and plated out over forty 96-well plates at 50 μL per well. The day after transfection, 150 μL of the CM63 medium containing the selective agent L-methionine sulphoximine (MSX) was added to each well to give a final MSX concentration of 50 μM. After approximately 3, 4 and 5 weeks of incubation the plates were screened for the presence of colonies using a cloning mirror. Any colonies identified were examined further using a microscope to evaluate if the colony had arisen from a single or multiple cells.

Two hundred and forty-five colonies were identified and screened for antibody production using an Octet based method. Antibody concentrations ranged from 0 to 172 μg/mL. Of the 245 cell lines screened, 214 were positive for antibody production. The cell lines were ranked based on productivity and the top 70 cell lines were selected. Cultures of these 70 cell lines were initially expanded in static culture and subsequently into suspension culture. Thirty cell lines that exhibited acceptable growth were selected and evaluated further in a batch shake-flask culture productivity screen. Cultures were gassed on days 4, 6, 8 and 10 and harvested on day 12. The concentration of antibody in the harvest supernatant samples was determined in HPLC using Protein A containing column. The antibody concentrations in the samples from the 30 cell lines ranged from 109 to 877 mg/L. The results of the batch shake-flask assessment were used to rank the cell lines, based on productivity. The top 20 cell lines were selected for further evaluation.

The growth and productivity data for these 20 selected cell lines expressing the ARGX-110 antibody was studied in fed-batch shake-flask cultures using CDACF medium and the results are shown in Table 24. Antibody concentrations at harvest ranged from 857 to 3922 mg/L, as determined on a Protein A HPLC column. Cell line F13 was selected to inoculate a disposable 10 L cell bag bioreactor. A harvest antibody concentration of 4327 mg/L was achieved. This cell line achieved an antibody concentration at harvest of 2711 mg/L in fed-batch shake-flask culture and had the second highest specific production rate (2.22 μg/cell/h) of all 6 cell lines assessed (B1, D4, D5, F1, F13 and F18). This cell line also exhibited acceptable growth characteristics.

TABLE 24 Summary of growth and productivity data for 20 selected cell lines, expressing the ARGX-110 antibody, grown in CDACF fed-batch shake-flask cultures. Maximum Via- Antibody Viable Cell bility Concen- Specific Concen- IVC at at tration at Production Cell tration Harvest⁽¹⁾ Harvest Harvest⁽²⁾ Rate (q_(p))⁽³⁾ Line (10⁶/mL) (10⁹cell.h/L) (%) (mg/L) (pg/cell/h) A1 8.55 1276 47.1 1536 1.204 A9 4.01 1039 71.5 1033 0.994 B1 8.60 1777 69.9 3922 2.206 B2 5.22 1277 59.7 2938 2.301 D4 9.10 1957 79.5 3346 1.709 D5 7.84 1791 78.5 3258 1.819 D12 7.76 1635 91.2 1424 0.871 D20 7.66 1525 60.6 3130 2.052 D24 4.25 973 28.3 1133 1.164 D30 7.58 1492 93.4 2095 1.405 F1 8.55 1717 41.0 3704 2.157 F13 6.14 1224 46.0 2711 2.215 F18 7.80 1641 50.0 3301 2.011 F20 7.76 1812 68.2 2095 1.156 F21 7.95 1693 39.7 2008 1.186 F22 8.13 1953 87.9 1572 0.805 F29 9.16 1944 88.3 1435 0.738 F31 7.27 1453 73.9 1540 1.060 F33 5.67 1409 7.2 857 0.608 F34 10.04 2049 93.4 1328 0.648 ⁽¹⁾Time integral of the viable cell concentration at harvest. ⁽²⁾Determined by Protein A HPLC. ⁽³⁾Calculated by linear regression analysis of the antibody concentration against the time integral of the viable cell concentration.

Example 18 Affinity of CD70 mAbs

18.1 Affinity for Cancer Cell Lines Expressing CD70

Several cancer cell lines were tested by FACS analysis for binding of ARGX-110 and SGN70 (described in US2010/0129362) at saturating mAb concentrations (at least 2 μg/ml). This was either done at 4° C. (1 hour incubation of cells with mAbs) or at 37° C. (15 minutes incubation of cells with mAbs). Binding was detected using anti-hIgG1-Fc-FITC (AF006, Binding Site) or anti-hIgG1-Fc-PE antibody (eBioscience, 12-4998). Fluorescence was measured using a flow cytometer. The results are summarized in Table 25. The third column of the table shows whether ARGX-110 binds with low (+), medium (++) or high (+++) affinity to the various cell lines tested.

In the right-hand column, the FACS signal (MFI) for ARGX-110 is divided by the FACS signal (MFI) for SGN70 for the experiment carried out at 37° C. These results demonstrate that ARGX-110 binds with higher affinity to the cells than SGN70, particularly for lower copy-number cell lines where it can be expected that high affinity binding of an antibody can be picked up more easily.

TABLE 25 Binding of CD70 mAbs ARGX-110 and SGN70 to cancer call lines Signal in FACS of ARGX-110/signal Type Cell line ARGX-110 in FACS of SGN70 Burkitt lymphoma Raji +++ 1.2 Large B cell lymphoma SU-DHL-6 + 2.4 Hodgkin lymphoma L428 +++ 1.4 Non Hodgkin lymphoma MHHPREB1 +++ 1.2 Mantle Cell Lymphoma Mino +++ 1.0 Jeko +++ Granta 519 ++ 0.9 Rec-1 + Chronic lymphocytic leukemia Mec1 +++ 1.2 JVM-3 ++ JVM-2 + 2.4 Cutaneous T cell lymphoma HUT78 +++ 1.0 HH + 1.7 Multiple Myeloma U266 +++ 0.8 JNN-3 +++ LP1 ++ AMO-1 +++ RPMI8226 +++ MM1.S ++ KMS11 +++ KMS12MB +++ Renal cell carcinoma 786-O +++ 2.1 Caki-1 ++ 1.9 A498 ++ 1.0 Astrocytoma U251 +++ Gastric carcinoma MKN-45 + 10.6 Lung carcinoma A549 + 3.8 EBC-1 + 8.9 Melanoma WM1205-Lu +++ WM852 + WM3248 +++ WM793 + WM1552C ++ WM115 +++ Glioblastoma GaMG + U87MG ++ 2.0 U343 ++ Ovarian carcinoma OAW-42 + SKOV3 + OVCAR3 + Pancreatic carcinoma PANC-1 ++ 2.9 PANC-89 +

In a further experiment, the apparent binding affinity of CD70 mAbs, ARGX-110, SGN70 and MDX1411 [see above], across a range of concentrations was determined for different cell lines. Cells were incubated for 1 hour at 4° C. with a dilution series of CD70 binding mAbs and binding was detected using anti-hIgG1-Fc-FITC or anti-hIgG-Fc-PE antibody. Fluorescence was measured using a flow cytometer and the median fluorescence was plotted. The results are shown in FIG. 15.

The results demonstrate that the affinity of the three different mAbs for CD70 on cells is comparable, but for some cell lines, SU-DHL-6, A549 and MKN45, binding of ARGX-110 is superior as compared to MDX1411 and SGN70. The EC50 for binding to SU-DHL-6, A549 and MKN45 is much higher for all mAbs as compared to EC50 on the other cell lines like U266 and many others, which probably indicates that the mAb is binding only with one arm (Fab), no longer allowing for avidity and thus resulting in lower affinities. Indeed, when tested in Biacore, the affinity of the Fab of ARGX-110 versus SGN70 and MDX1411 is much higher (see below).

18.2 Affinity for Patient Cells Expressing CD70

Primary cells taken from chronic lymphocytic leukaemia (CLL) patients (2 high risk patients, 1 low risk patient) were plated at 250,000 cells/well in round bottom 96 well plates in RPMI 1640+10% FBS and mAbs at a concentration of 5 μg/ml in RPMI+10% FBS were added. After incubation for up to 5 hours at 37° C., cells were washed twice with ice-cold PBS. Alexa Fluor 647 labelled goat Anti-Human IgG (Invitrogen Cat#21445) was diluted 1/500 in PBS/1% BSA and incubated for 20 minutes at 4° C. The plate was washed twice with ice-cold PBS and 4% paraformaldehyde was added for 15 minutes at room temperature to fix the cells. Signals were measured by FACS. The results are shown in FIG. 16. The results demonstrate that ARGX-110 binds well to the cells from the CLL patients whereas SGN70 almost gave no binding signals.

18.3 Affinity of CD70 Fabs for Recombinant CD70 in Biacore

Recombinant human CD70 was immobilized on a CM5 Biacore chip. The immobilization was performed in accordance with a method provided by Biacore and by using the NHS/EDC kit (Biacore AB): after activation of the chip, a solution of 50 μg/ml of recombinant CD70 in 10 mM acetate buffer with pH of 5 was prepared and 1 μl of this solution (50 ng) was injected resulting in a surface density of approximately 1000 RU.

Fabs were prepared for the CD70 mAbs ARGX-110, MDX1411 and SGN70 by papain digestion. These Fabs, at a concentration of approximately 100-400 μg/ml, were diluted 6-fold in HEPES-buffered saline (0.1M HEPES, 1.5M NaCl, 30 mM EDTA, 0.5% v/v surfactant P20). They were injected (30 μl) and passed through the flow cells at a flow rate of 30 μl/min. After binding of the Fab to CD70, the off-rate was monitored for a period of 10 minutes. After dissociation, the flow cell surfaces were regenerated by injecting 5 μl of 10 mM NaOH. Sometimes multiple injections of NaOH were needed to regenerate the surfaces depending on the affinity of the Fabs. Off-rate analysis was done by applying the BIAevaluation software. First, the sensogram of the blank runs were subtracted from those obtained with the coated flow cell. Then the off-rate was determined for a time range of 10 minutes using the Fit kinetics application and the Kd value was calculated. The off rates are summarized in Table 26.

TABLE 26 “off rate” of Fabs for binding to human CD70 Off rate [10⁻⁴ s⁻¹] ARGX-110 1.4 SGN70 7 MDX1411 17 18.4 Spiking Experiments to Assess Lysis of SU-DHL-6 Cells Bound by CD70 mAbs

SU-DHL-6 cells (a diffuse large B cell lymphoma cell line) were pipeted into wells at 50,000 cells/well which were spiked then into 300,000 freshly isolated PBMCs/well from healthy donors and mAbs were added at different concentrations. The samples were incubated for 2 days and analysed by FACS for the depletion of the cell line (as measured with anti-human CD19 APC (eBioscience 17-0199-42)). The best potency in cell killing was obtained with ARGX-110, which also gives the highest % of lysis. The results are shown in FIG. 17.

Example 19 CD27-CD70 Blocking Activity of CD70 mAbs

The interaction between CD70 and CD27 may contribute to tumour cell survival, proliferation and/or immune suppression within the tumour microenvironment. The ability of CD70 mAbs to block the interaction between CD70 and CD27 was therefore assessed by ELISA.

In this assay, a microtiter plate (Nunc Maxisorb) was first coated with 100 μl anti-FLAG M2 monoclonal antibody (Sigma Aldrich, F3165) at 1250-fold dilution in PBS (to achieve a final concentration of approximately 3.5 μg/ml) overnight at 4° C. The plate was washed once with PBS-Tween and incubated at RT for 2 hours with 300 μl PBS-1% casein. The plate was washed a further three times with PBS-Tween. 100 μl of 5 ng/ml (80 pM) Flag-TNC-CD70 (The Journal of Immunology, 2009, 183: 1851-1861) diluted in PBS-0.1% casein was added to the plate and incubated at RT for 1 hour while shaking. The plate was washed five times with PBS-Tween. The CD70 mAb to be tested was added to the plate; various concentrations were achieved by diluting the stock antibody solution in PBS-0.1% casein. Immediately thereafter, 50 μl of 1 μg/ml (final concentration 6.5 nM) recombinant human CD27 Fc chimera (R&D systems 382-CD, MW=46.5 kDa) was added, and incubated at RT for 1 hour while shaking. The plate was washed five times with PBS Tween. 100 μl of biotinylated anti-CD27 (eBioscience 13-0271), diluted 500-fold in PBS-0.1% casein, was added, and the plate incubated at RT for a further hour, while shaking. The plate was washed five times with PBS Tween. 100 μl of Strep-HRP (Jackson Immunoresearch 016-030-084) diluted 5000 fold in PBS-0.1% casein was added, and the plate incubated at RT for a further hour, while shaking. The plate was washed five times with PBS-Tween. 100 μl TMB was added to the plate and the OD at 620 nm was measured.

Using this assay, the blocking potency of ARGX-110 was compared to the potency of SGN70 and MDX1411. As shown in FIG. 18, ARGX-110 is much more potent (about 100-fold) in blocking the interaction between CD70 and CD27 than the two benchmark mAbs (IC50 ARGX-110=67 ng/ml, MDX1411=5500 ng/ml and SGN70=4972 ng/ml).

Example 20 Binding Properties of CD70 mAbs

20.1 Cross-reactivity with CD70 of Non-human Species

Determining the animal cross-reactivity of CD70 mAbs is useful for the purposes of assessing which animal models can be used for in vivo proof of concept studies, and which species are most suited for toxicology studies. Rhesus CD70 is 94% identical to human CD70 and Cynomolgus monkey CD70 is 95% identical to human CD70. An alignment of the sequences from the different species is shown in FIG. 19. The mouse and rat CD70 sequences are also included so as to highlight the differences as compared with the human sequence.

CD70 mAbs ARGX-110, SGN70 and MDX1411 were tested for binding to U266 (a human multiple myeloma cell line), LCL8664 cells (rhesus B cell lymphoma cell line, ATCC-CRL-1805) and to HSC-F cells (cynomolgus monkey T-cell line, fetal spleen-derived lymphocytes, Japanese health science foundation) cells in a dilution series starting at a concentration of 10-20 μg/ml.

Detection was performed using goat anti human IgG1 FITC. Samples were analysed by FACS. The results are shown in FIG. 20. The copy-number of CD70 on both monkey cell lines is very low (low signals in FACS). However, ARGX-110 has a high affinity for cynomolgus and rhesus CD70 as compared to the affinity of SGN70 and MDX1411 for CD70 of these species.

20.2 Blocking Potency of CD70 mAbs

The ability of CD70 mAbs (ARGX-110, SGN70 and MDX1411) to block the interaction between CD70 and CD27 was tested using the blocking ELISA described in Example 19 above. The assay was adapted such to measure the blocking potency of each antibody for human, rhesus and cynomolgus monkey Flag-TNC-CD70 (Wyzgol, et al., J. Immunol., 2009, 183: 1851-1861).

In the ELISA, exactly the same conditions were used for CD70 from the different species so that the IC50 values between the different species could be compared directly. The results are shown in FIG. 21 and support the results seen using cells from humans, rhesus monkeys and cynomolgus monkeys. ARGX-110 has an unaltered potency and therefore an unaltered affinity for CD70 of monkeys as compared to human, whereas SGN70 and especially MDX1411 have a lower affinity for monkey than for human CD70. IC50 values are summarized in Table 27.

TABLE 27 IC50 for blocking of the CD27-CD70 interaction in ELISA for CD70 from different species IC50 [ng/ml] in inhibition ELISA Rhesus Cynomolgus Cells Human monkey monkey ARGX-110 39 75 48 SGN70 54 178 112 MDX1411 44 >10000 >10000 20.3 Binding to Denatured CD70

The binding of CD70 mAbs to denatured recombinant CD70 was assessed by ELISA. Recombinant CD70 was denatured by heating for 5 minutes at 95° C., followed by immediate cooling on ice for 5 minutes. A microtiter plate was coated with 5 μg/ml CD70 with or without denaturation. The plate was blocked and the CD70 mAbs for testing were applied at 10 μg/ml. After washing, binding of the mAbs was detected using anti-human IgG-HRP. As a positive control for the coating, instead of using the CD70 mAbs, an anti-Flag mouse-derived mAb was applied and detection was with anti-mouse-HRP. TMB was used as a substrate and OD at 620 nm was measured. The results from this ELISA are shown in FIG. 22. The results demonstrate that ARGX-110 binds to a different epitope than SGN70, MDX1411 (MDX2H5) and MDX69A7 as ARGX-110 still binds to denatured CD70 whereas the other mAbs don't. Amongst the other mAbs, some are also binding to denatured CD70 (fe 5F4, 9G2, 57B6).

20.4 Epitope Mapping Using Mouse-Human Chimeras

Human-mouse CD70 ECD fusion proteins were constructed by exchanging domains of human and mouse CD70 in order to map the domain recognition of the mAbs. The construction was done using standard recombinant DNA and PCR methodologies. The mouse and human chimeras were cloned into a eukaryote expression vector with a Flag tag for capturing in ELISA and a TNC for trimerisation of the protein (Flag-TNC-CD70). Proteins were expressed as soluble proteins in HEK293 cells. The sequence of the different chimeras is shown in FIG. 23.

A Maxisorb (Nunc) microtiter plate was coated with 3.5 μg/ml mouse M2 anti-Flag mAb (Sigma Aldrich, F3165) and the different chimeras were captured. A dilution series of the CD70 mAbs to be tested was applied starting at a concentration of 10 μg/ml and making 3-fold dilutions. After 2 hours, binding was detected using anti-human-Fc-HRP at a 16000-fold dilution. Staining was done with ABTS and OD at 405 nm was measured. As a positive control, CD27-Fc was applied which is able to bind to both human and mouse CD70 with good affinity as well as to the chimeric human-mouse CD70 variants. The EC50 values for binding are summarized in Table 28.

TABLE 28 Binding of ARGX-1 10 to human-mouse CD70 chimeras shown in FIG. 23. EC50 [ng/ml] ARGX-110 CD27 human 22 79 chim-1 27 159 chim-2.1 15 51 chim-2.2 16 56 chim-2.3 15 chim-2.4 14 76 chim-2 NB 99 chim-3 NB 94 chim-4 NB 51 mouse NB 41 NB = no binding

The results demonstrate that ARGX-110 can bind to human Flag-TNC-CD70 and to chimeras 1, 2.1, 2.2, 2.3 and 2.4 but does not bind to chimeras 2, 3 and 4 and mouse Flag-TNC-CD70. This indicates that the epitope for ARGX-110 is within the following amino acid sequence: HIQVTLAICSS (SEQ ID NO:342).

Example 21 CD70 Internalization of Different Tumor Cell Lines and Primary Tumor Cells

It has been demonstrated that binding of CD70 antibodies to the renal carcinoma derived cell lines 786-O and A498 results in the rapid (within 1 hour) internalization of the antibody-receptor complex (Adam et al., Br. J. Cancer (2006) 95: 298-306). In order to test for internalization of mAbs ARGX-110, SGN70 and MDX1411, 786-O cells were cultured in a 96-well microtiter plate and incubated overnight at 37° C. 2.5 μg/ml mAb was added and incubated with the cells for 0-24 hours at 37° C. Plates were washed 3 times 5′ with stripping buffer (150 mM NaCl, 100 mM Glycine, pH=2.5; coded “IN” representing the amount of mAb internalized via CD70) or PBS (coded “OUT” and representing the amount of mAb bound to the receptor at the outside of the cell). Subsequently, cells were fixed with 4% paraformaldehyde for 30′ at RT, washed with PBS, and incubated 5′ with 0.2% Triton-X-100 (“IN”) or PBS (“OUT”). Next, cells were washed twice and incubated at RT for 10′ with 100 mM glycine followed by 30 mins incubation with PBS+1% BSA. Finally cells were stained with goat anti-human Fc (Jackson immunoresearch 109-005-098) and anti-goat IRDYE800 (L1-cor 926-32214) before analysis on the L1-Cor Odyssey infrared scanner. The % of mAb OUT as a function of time is shown in FIG. 24.

The results demonstrate that internalization for all mAbs is comparable and that none of the mAbs internalizes completely. Initially, between 0 and 2 hours, the mAb internalization goes very fast, but then it seems that a steady state is reached where about 30% of the mAb remains at the outside of the cell, even after 24 hours of incubation at 37° C.

The internalization of CD70-bound mAbs was also assessed using other cell lines. In these experiments, only the signal of the mAb outside the cell was measured as a function of time using FACS analysis according to the protocol described below. This alternative protocol was determined to be a reliable readout as compared with the method described above (see FIG. 24, right-hand panel).

For suspension cells, cells were centrifuged, counted and the pellet resuspended in medium to a density of 5×10⁵ cells/ml, and 100 μl of this suspension was added per well of a 96-well V-bottom plate. For adherent cells, cells were seeded in a 96-well plate and grown ON at 37° C., 5% CO₂ until cells were fully attached. Cells were plated at a density which allowed linear cell growth for an additional 48 h period.

The mAbs for testing were diluted and incubated with the cells at 37° C., 5% CO₂ for varying time periods: 24 h, 8 h, 6 h, 4 h, 2 h, 1 h, 30 min, 15 min, 5 min, 0 min (=no mAb). Following incubation, the plates were washed on ice with cold FACS buffer to stop the internalization reaction. At this stage, adherent cells were detached from the plate using cell dissociation solution (Sigma) and transferred to a V-bottom 96-well plate. Next, cells were spun down at 4° C. The supernatant was removed by gently inverting the plate. The cells were washed twice by gently re-suspending the cell pellet in 100 μl cold FACS buffer. After washing, the cell pellet was resuspended in 100 μl anti-hu IgG-FITC, diluted 1/500 in FACS buffer. The plate was incubated for 30 min at 4° C., while shaking, and the cells were washed a further three times with cold FACS buffer. The cells were resuspended in 100 μl FACS buffer and fluorescence was measured immediately. The median mean fluorescence intensity (MFI) was plotted versus time.

Several of the experiments were repeated two or more times. Results from two experiments were very comparable. In some experiments, using U266, SU-DHL-6 and Raji cells, a concentration range (20 ng/ml-5 μg/ml) was used to see if there was an effect of mAb concentration on internalization rate. No such effect was observed (data not shown).

The results for different cell lines tested are shown in Table 29 and demonstrate that internalization is not a common phenomenon, actually it is quite a rare event. Table 29 summarizes the percentage of internalization after 6 hours for the different cell lines. These results show that most of ARGX-110 remains bound to the outside of the cells making the cells more susceptible to ADCC, CDC and ADCP.

TABLE 29 % internalization of CD70 mAbs on different cell lines after 6 hours. % internalization Average after 6 % per Type Cell line hours indication Burkitt lymphoma Raji 2 2 Large B cell lymphoma SU-DHL-6 3 3 Hodgkin lymphoma L428 15 15 Non Hodgkin lymphoma MHHPREB1 19 19 Mantle Cell Lymphoma Mino 12 7 Granta 519 7 Rec-1 2 Chronic lymphocytic Mec1 3 7 leukemia JVM-2 8 JVM-3 11 Patient HR 5 Patient HR 7 Patient LR 8 Cutaneous T cell HUT78 35 33 lymphoma HH 31 Multiple Myeloma U266 38 AMO-1 36 RPMI8226 38 34 MM1.S 35 KMS12MB 49 Renal cell carcinoma 786-O 58 54 Caki-1 37 A498 66 Astrocytoma U251 52 52 Gastric carcinoma MKN-45 0 0 Lung carcinoma A549 0 3 EBC-1 6 Melanoma WM1205-Lu 45 44 WM3248 34 WM115 52 Glioblastoma U87MG 10 21 U343 32 Ovarian carcinoma SKOV3 51 51 Pancreatic carcinoma PANC-1 29 25 PANC-89 20

TABLE 30 nucleotide sequences encoding selected CD70 antibodies mAb 41D12 Heavy chain gccgccaccATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCAC SEQ ID NO: CGCCACAGGCGTCCACTCT GAGGTGCAGCTCGTGGAGTCTGGG 344 GGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTG CAGCCTCTGGATTCACCTTCAGTGTCTACTACATGAACTGGGTC CGCCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCAGATATTA ATAATGAAGGTGGTACTACATACTATGCAGACTCCGTGAAGGGC CGATTCACCATCTCCAGAGACAACTCTAAGAACAGCCTGTATCT GCAAATGAACAGCCTGCGCGCCGAGGACACGGCCGTGTACTAC TGCGCGAGAGATGCCGGATATAGCAACCATGTACCCATCTTTGA TTCTTGGGGCCAGGGGACCCTGGTCACTGTCTCCTCAGCCAGTA CAAAAGGTCCAAGTGTGTTCCCTCTTGCTCCCTCATCCAAGAGTA CCAGTGGAGGCACCGCCGCTCTTGGCTGCTTGGTTAAGGATTAT TTCCCAGAGCCTGTCACTGTTTCATGGAACTCCGGCGCCTTGACA TCTGGTGTGCATACCTTTCCAGCCGTGCTGCAGTCAAGTGGCCTC TACAGCCTCAGTAGCGTGGTCACTGTGCCCAGCAGCTCTCTCGG CACACAAACTTATATCTGTAATGTGAATCATAAGCCTTCAAATACC AAGGTGGATAAGAAAGTGGAACCAAAATCATGTGACAAGACACAC ACCTGCCCTCCTTGTCCAGCCCCCGAACTGCTGGGTGGGCCCAG CGTGTTCCTGTTTCCTCCTAAACCCAAAGACACTCTGATGATTAGT AGGACCCCAGAAGTCACTTGCGTGGTGGTTGACGTGTCACATGA AGATCCCGAGGTCAAGTTCAATTGGTATGTTGACGGGGTCGAAGT TCACAACGCTAAAACTAAACCAAGAGAGGAACAGTATAACTCTAC CTACCGGGTGGTGAGTGTTCTGACTGTCCTCCATCAAGACTGGCT GAATGGCAAAGAATACAAGTGTAAGGTGAGCAACAAAGCCCTGC CCGCTCCTATAGAGAAAACAATATCCAAAGCCAAAGGTCAACCTC GCGAGCCACAGGTGTACACCCTCCCACCAAGCCGCGATGAACTT ACTAAGAACCAAGTCTCTCTTACTTGCCTGGTTAAGGGGTTCTAT CCATCCGACATTGCAGTCGAGTGGGAGTCTAATGGACAGCCTGA GAACAACTACAAAACCACCCCTCCTGTTCTGGATTCTGACGGATC TTTCTTCCTTTATTCTAAACTCACCGTGGATAAAAGCAGGTGGCAG CAGGGCAACGTGTTCAGCTGTTCCGTTATGCATGAGGCCCTGCA TAACCATTATACCCAGAAGTCTTTGTCCCTCAGTCCAGGAAAGTG A kozak LEADER VARIABLE REGION LAMBDA CONSTANT REGION Light chain gccgccaccATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCAC SEQ ID NO: CGCCACAGGCGTCCACTCT CAGGCAGTGGTGACCCAGGAGCCT 345 TCCCTGACAGTGTCTCCAGGAGGGACGGTCACGCTCACCTGCG GCCTCAAATCTGGGTCTGTCACTTCCGATAACTTCCCCACTTGGT ACCAGCAGACACCAGGCCAGGCTCCCCGATTGCTTATCTACAA CACAAACACCCGTCACTCTGGCGTCCCCGACCGCTTCTCCGGAT CCATCCTGGGCAACAAAGCCGCCCTCACCATCACGGGGGCCCA GGCCGACGACGAGGCCGAATATTTCTGTGCTCTGTTCATAAGTA ATCCTAGTGTTGAGTTCGGCGGAGGGACCCAACTGACCGTCCTA GGTCAACCTAAAGCAGCACCTTCAGTTACTCTGTTTCCACCTAGT TCAGAGGAACTGCAGGCCAATAAAGCCACACTCGTCTGCCTCAT CAGTGACTTCTACCCAGGAGCCGTGACCGTGGCCTGGAAAGCCG ACAGTAGCCCCGTGAAGGCCGGGGTGGAGACAACAACTCCTAGT AAACAGAGTAATAACAAATATGCCGCTAGTAGTTATCTCTCCCTCA CTCCCGAGCAGTGGAAGTCTCACAGAAGTTACTCTTGTCAGGTTA CTCACGAGGGTTCCACAGTGGAAAAGACTGTGGCCCCTACTGAA TGTAGTTGA kozak LEADER VARIABLE REGION LAMBDA CONSTANT REGION 57B6 Heavy chain CAGTTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTG SEQ ID NO: GGGGGTCTCTGAGACTC 346 TCTTGTGCAGCCTCTGGATTCAGCTTCAGTCACTATGCCATGAGC TGGGTCCGCCAGGCT CCAGGAAAGGGGCTAGAGTGGGTCTCAGGTGATAATACCTACGA TGGTGGTACAAGGTAT CAAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGG CAAGAACACGCTGTAT CTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTGTATTA CTGTGCAAAAGATACT GGTAGAGGCATTATGGGGGAGTACGGCATGGACTACTGGGGCAA AGGGACCCTGGTCACC GTCTCCTCA Light chain CAGACTGTGGTGACCCAGGAGCCGTCCCTGTCAGTGTCTCCAGG SEQ ID NO: AGGGACGGTCACACTC 347 ACCTGCGGCCTCAAGTCTGGGTCTGTCACTTCCAGTAACTACCCT GCTTGGTACCAGCAG ACACCAGGCCAGGCTCCCCGATTGCTTATCTACAACACAAACAGC CGTCACTCTGGGGTC CCCAGTCGCTTCTCCGGATCCATCTCTGGGAACAAAGCCGCCCT CACCATCACGGGGGCC CAGCCCGAGGACGAGGCCGACTATTACTGTGCTCTGTACATGGG TAGTGGTAGTGCCAAT GCTATGTTCGGCGGAGGGACCCATCTGACCGTCCTGGGTCA 59D10 Heavy chain GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTG SEQ ID NO: GGGGGTCTCTGAGACTC 348 TCCTGTGCAGCGTCCGAATTGTCCTTCAGTATTTCTGAGATGACC TGGGTCCGCCAGGCT CCAGGAAAGGGGCTCGAGTGGGTCTCAGGTATTAGTGGTGTAAC TGGTGGTAGTAGTACA AGTTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGA CAACGACAAGAACACG TTGTATCTACAAATGAACAGCCTGATACCCGAGGACACGGCCGTA TATTACTGTGCAACA ACTAGTGGTACTTACTACTTCATCCCCGAGTATGAGTACTGGGGC CAGGGGACCCAGGTC ACCGTCTCCTCA Light chain CAGTCTGTGCTGACCCAGCCTCCCTCCGTGTCTGGGTCTCCAGG SEQ ID NO: AAAGACGGTCACCATC 349 TCCTGTGCAGGAACCAGCAGTGATGTTGGGTATGGATACTATGTC TCCTGGTATCAACAG TTCCCAGGAATGGCCCCCAAACTCCTGATATATGACGTCAATAAA CGGGCCTCAGGGATC GCTGATCGCTTCTCTGGCTCCAAGGCCGGCAACACTGCCTCCCT GACCATCTCTGGGCTC CAGTCTGAGGACGAGGCTGATTATTACTGTGCCTCATATAGAAGT AGCGCCAATGCTGTG TTCGGCGGAGGGACCCATCTGACCGTCCTGGGT 

The invention claimed is:
 1. An isolated polynucleotide that encodes a monoclonal antibody, or a fragment thereof, which specifically binds to an epitope of human CD70, wherein the epitope is within the amino acid sequence HIQVTLAICSS (SEQ ID NO:342), wherein the monoclonal antibody or fragment thereof, when tested as a Fab fragment, has an off-rate (k_(off)) as measured by surface plasmon resonance for human CD70 of less than 7×10⁻⁴ s^(−1.)
 2. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein the VH and VL domains, or one or more complementarity-determining regions (CDRs) thereof, are derived from a camelid species.
 3. An isolated polynucleotide that encodes a monoclonal antibody, or a fragment thereof, which specifically binds to human CD70, said monoclonal antibody or fragment thereof comprising a heavy chain variable (VH) domain comprising CDR3 (HCDR3), CDR2 (HCDR2), and CDR1 (HCDR1), regions, and a light chain variable (VL) domain comprising CDR3 (LCDR3), CDR2 (LCDR2), and CDR1 (LCDR1) regions, wherein the HCDR3, HCDR2, HCDR1, LCDR3, LCDR2, and LCDR1 regions comprise the amino acid sequence set forth in SEQ ID NO:50, SEQ ID NO:27, SEQ ID NO:11, SEQ ID NO:160, SEQ ID NO:119, and SEQ ID NO:250, respectively.
 4. The isolated polynucleotide of claim 3, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:223, and the VL domain comprises the amino acid sequence of SEQ ID NO:241.
 5. An isolated polynucleotide that encodes a monoclonal antibody, or a fragment thereof, which specifically binds to human CD70, said monoclonal antibody or fragment thereof comprising a heavy chain variable (VH) domain comprising CDR3 (HCDR3), CDR2 (HCDR2), and CDR1 (HCDR1) regions, and a light chain variable (VL) domain compising CDR3 (LCDR3), CDR2 (LCDR2), and CDR1 (LCDR1) regions, wherein the HCDR3 region comprises the amino acid sequence of SEQ ID NO:50; the HCDR2 region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:27 and SEQ ID NO:249; and the HCDR1 region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:11 and SEQ ID NO:248.
 6. The isolated polynucleotide of claim 5, wherein the HCDR3 region comprises SEQ ID NO:50; the HCDR2 region comprises SEQ ID NO:27; and the HCDR1 region comprises SEQ ID NO:11.
 7. The isolated polynucleotide of claim 5, wherein the LCDR3 region comprises SEQ ID NO:160; the LCDR2 region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:119 and SEQ ID NO:110; and the LCDR1 region comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:87, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, and SEQ ID NO:253.
 8. The isolated polynucleotide of claim 7, wherein the LCDR3 region comprises SEQ ID NO:160; the LCDR2 region comprises SEQ ID NO:119; and the LCDR1 region comprises SEQ ID NO:250.
 9. The isolated polynucleotide of claim 8, wherein the HCDR3 region consists of SEQ ID NO:50; the HCDR2 region consists of SEQ ID NO:27; the HCDR1 region consists of SEQ ID NO:11; the LCDR3 region consists of SEQ ID NO:160; the HCDR2 region consists of SEQ ID NO:119; and the HCDR1 region consists of SEQ ID NO:250.
 10. The isolated polynucleotide of claim 3, wherein the VH domain consists of the amino acid sequence shown as SEQ ID NO:223, and the VL domain consist of the amino acid sequence shown as SEQ ID NO:241.
 11. An isolated polynucleotide that encodes a monoclonal antibody, or a fragment thereof, which specifically binds to human CD70, said monoclonal antibody or fragment thereof comprising: a) a heavy chain variable (VH) domain with the amino acid sequence set forth in SEQ ID NO:178 comprising one or more amino acid substitutions at Kabat positions selected from the group consisting of H6, H18, H24, H31, H56, H74, H77, H79, H83, H84, H89, H93, H94, H108, H110, and H112; and b) a light chain variable (VL) domain with the amino acid sequence set forth in SEQ ID NO:201 comprising one or more amino acid substitutions at Kabat positions selected from the group consisting of L2, L11, L12, L25, L26, L30, L46, L53, L60, L61, L67, L68, L76, L80, L81, L85, and L87, wherein the thermal stability of the antibody, or the fragment thereof, is greater than the thermal stability of an antibody, or antigen binding fragment thereof, comprising a heavy chain variable domain with the amino acid sequence set forth in SEQ ID NO:178 and a light chain variable domain with the amino acid sequence set forth in SEQ ID NO:201.
 12. The isolated polynucleotide encoding the monoclonal antibody according to claim 11 which is a llama-human chimeric antibody, wherein the VH and VL domains, or one or more CDRs thereof are derived from llama, and one or more of the constant domains are derived from a human immunoglobulin.
 13. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof, when tested as a Fab fragment, has an off-rate (k_(off)) as measured by surface plasmon resonance for human CD70 of less than or equal to 4.8×10⁻⁴ s⁻¹.
 14. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof is a chimeric antibody or a humanized antibody.
 15. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof is a chimeric antibody, wherein the chimeric antibody comprises a constant domain derived from a human immunoglobulin.
 16. The isolated polynucleotide according to claim 15, wherein the constant domain is derived from a human immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
 17. The isolated polynucleotide according to claim 15, wherein the constant domain is derived from human IgG1.
 18. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof is a llama-human chimeric antibody comprising VH and VL domains, or one or more CDRs thereof, derived from llama, and one or more constant domains derived from a human immunoglobulin.
 19. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof is capable of directing immune effector function against a cell expressing human CD70 on its surface, wherein the immune effector function is selected from the group consisting of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and any combination thereof.
 20. The isolated polynucleotide of claim 1, wherein the monoclonal antibody or fragment thereof is an scFc, a F(ab′)₂ fragment, a Fab fragment, a Fab′ fragment, an Fd fragment, an Fv fragment, or a single domain antibody (Dab) fragment.
 21. The isolated polynucleotide of claim 3, wherein the VH domain comprises the amino acid sequence of SEQ ID NO:223.
 22. The isolated polynucleotide of claim 3, wherein VL domain comprises the amino acid sequence of SEQ ID NO:241. 